48120, 23479 and 46689, novel human hydrolases and uses thereof

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 23479, 48120, and 46689 nucleic acid molecules, which encode novel hydrolases. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 23479, 48120, and 46689 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 23479, 48120, or 46689 gene has been introduced or disrupted. The invention still further provides isolated 23479, 48120, and 46689 proteins, fusion proteins, antigenic peptides and anti-23479, 48120, or 46689 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applications 60/237,991, filed on Oct. 5, 2000, and 60/238,170, filed on Oct. 5, 2000, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Hydrolases are a large class of enzymes that use a water molecule to catalyze the cleavage of a chemical bond. Hydrolases play important roles in the synthesis and breakdown of almost all major metabolic intermediates, including polypeptides, nucleic acids, and lipids.

[0003] One family of hydrolases consists of the ubiquitin carboxy-terminal hydrolases. The ubiquitin pathway is one example of a post-translational mechanism used to regulate protein levels. Ubiquitin is a highly conserved polypeptide expressed in all eukaryotic cells that marks proteins for degradation. Ubiquitin is attached as a single molecule or as a conjugated form to lysine residue(s) of proteins via formation of an isopeptide bond at the C-terminal glycine residue. Most ubiquitinated proteins are subsequently targeted to the 26S proteasome, a multicatalytic protease, which cleaves the marked protein into peptide fragments.

[0004] Only the protein conjugated to ubiquitin is degraded via the proteasome; ubiquitin itself is recycled by ubiquitin carboxy-terminal hydrolases (UCH; sometimes abbreviated UCTH), which cleave the bond between ubiquitin and the protein targeted for degradation. These enzymes constitute a family of thiol proteases, and homologues have been found in, for example, yeast (Miller et al., BioTechnology 7:698-704, 1989; Tobias and Varshavsky, J. Biol. Chem. 266:12021-12028, 1991; Baker et al., J. Biol. Chem. 267:23364-23375, 1992), bovine (Papa and Hochstrasser, Nature 366:313-319, 1993), avian (Woo et al., J. Biol. Chem. 270:18766-18773, 1995), Drosophila (Zhang et al., Dev. Biol. 17:214, 1993) and human (Wilkinson et al., Science 246:670, 1989) cells.

[0005] Another family of hydorlases consists of α/β hydrolases. The α/β hydrolase family of enzymes is a phylogenetically diverse group of enzymes that share a common fold, typically comprising an eight-stranded J-sheet surrounded by I-helices (Ollis, D. et al. (1992) Protein Eng 5:197-211; Nardini and Dikkstra (1999) Curr Opin Str Bio 9:732-737). Members of the α/β hydrolase family are found in nearly all organisms, from microbes to plants to humans. Enzymes possessing the α/β hydrolase fold diverged from a common ancestor, but have preserved a catalytic mechanism that utilizes a triad of residues consisting of a nucleophile, an acidic residue, and a histidine residue (Ollis, D. et al. (1992) Protein Eng. 5:197-211). Although only the histidine residue is invariant, the other two residues in the triad are limited in terms of the amino acid residues that are functionally acceptable. Thus, the nucleophile is usually a serine residue, but can also be an aspartate or a cysteine residue, while the acidic residue is either an aspartic or glutamic acid residue (Schrag, J. et al. (1997) Meth Enzymol 284:85-107). The relative order of the three catalytic residues in the amino acid chain is always the same: nucleophile, acid, histidine.

[0006] Members of the α/β hydrolase family of enzymes include enzymes that hydrolyze ester bonds (e.g., phosphatases, sulfatases, exonucleases, and endonucleases), glycosidases, enzymes that act on ether bonds, peptidases (e.g., exopeptidases and endopeptidases), as well as enzymes that hydrolyze carbon-nitrogen bonds, acid anhydrides, carbon-carbon bonds, halide bonds, phosphorous-nitrogen bonds, sulfur-nitrogen bonds, carbon-phosphorous bonds, and sulfur-sulfur bonds (E. C. Webb ed., Enzyme Nomenclature, pp. 306-450, ©1992 Academic Press, Inc. San Diego, Calif.). Some specific biological activities of these enzymes include lipase activity, e.g., fungal, bacterial and pancreatic lipases, acetylcholinesterase activity, serine carboxypeptidase activity, prolyl aminopeptidase activity, haloalkane dehalogenase activity, dienelactone hydrolase activity, A2 bromoperoxidase activity, and thioesterase activity (Schrag, J. et al., supra). Acetylcholinesterases, epoxide hydrolases, cholesterol esterases, and lipases have particular medical importance. Inhibitors of acetylcholinesterase are useful therapeutic agents for the treatment of Alzheimer's disease, myasthenia gravis, and glaucoma; epoxide hydrolases and dienelactone hydrolases detoxify harmful aromatic compounds in mammals; and the human hormone sensitive lipase catalyzes the rate-limiting reaction of fat hydrolysis in adipocytes.

[0007] Given the important role that hydrolases play in the synthesis and breakdown of metabolic intermediates, including polypeptides, nucleic acids, and lipids, it is not surprising that their activity significantly impacts the activity of the cell. For example, hydrolases contribute to the growth and differentiation of the cell, to cellular proliferation, adhesion, and motility, and to the interaction and communication that takes place between cells. In addition, hydrolases are important in the conversion of pro-proteins and pro-hormones to their active forms, the inactivation of peptides, the biotransformation of compounds (e.g., a toxin or carcinogen), antigen presentation, and the regulation of synaptic transmission.

SUMMARY OF THE INVENTION

[0008] The present invention is based, in part, on the discovery of novel hydorlase molecules, referred to herein as “23479, 48120, and 46689”. The nucleotide sequence of cDNAs encoding 23479, 48120, and 46689 are recited in SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 7, respectively, and the amino acid sequences of 23479, 48120, and 46689 polypeptides are recited in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, SEQ ID NO: 5, and SEQ ID NO: 8, respectively. In addition, the nucleotide sequences of the coding regions are recited in SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9, respectively.

[0009] Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 23479, 48120, or 46689 protein or polypeptide, e.g., a biologically active portion of the 23479, 48120, or 46689 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In other embodiments, the invention provides isolated 23479, 48120, or 46689 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, or the sequence of a DNA insert of the plasmids deposited with ATCC Accession Numbers as described herein. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, or the sequence of a DNA insert of the plasmids deposited with ATCC Accession Numbers as described herein. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, or the sequence of a DNA insert of the plasmids deposited with ATCC Accession Numbers as described herein, wherein the nucleic acid encodes a full length 23479, 48120, or 46689 protein or an active fragment thereof.

[0010] In a related aspect, the invention further provides nucleic acid constructs that include a 23479, 48120, or 46689 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 23479, 48120, or 46689 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 23479, 48120, or 46689 nucleic acid molecules and polypeptides.

[0011] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 23479, 48120, or 46689-encoding nucleic acids.

[0012] In still another related aspect, isolated nucleic acid molecules that are antisense to a 23479, 48120, or 46689-encoding nucleic acid molecule are provided.

[0013] In another aspect, the invention features, 23479, 48120, or 46689 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 23479, 48120, or 46689-mediated or -related disorders. In another embodiment, the invention provides 23479, 48120, or 46689 polypeptides having a 23479, 48120, or 46689 activity. Preferred polypeptides are 23479, 48120, or 46689 proteins including at least one hydrolase domain, and, preferably, having a 23479, 48120, or 46689 activity, e.g., a 23479, 48120, or 46689 activity as described herein.

[0014] In other embodiments, the invention provides 23479, 48120, or 46689 polypeptides, e.g., a 23479, 48120, or 46689 polypeptide having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or an amino acid sequence encoded by a cDNA insert of one of the plasmids deposited with ATCC Accession Number as described herein; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or an amino acid sequence encoded by a cDNA insert of one of the plasmids deposited with ATCC Accession Number as described herein; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, or the sequence of a DNA insert of the plasmids deposited with ATCC Accession Numbers as described herein, wherein the nucleic acid encodes a full length 23479, 48120, or 46689 protein or an active fragment thereof.

[0015] In a related aspect, the invention provides 23479, 48120, or 46689 polypeptides or fragments operatively linked to non-23479, 48120, or 46689 polypeptides to form fusion proteins.

[0016] In another aspect, the invention features antibodies and antigen-binding fragments thereof that react with or, more preferably, specifically bind 23479, 48120, or 46689 polypeptides or fragments thereof, e.g., a hydrolase domain of a 23479, 48120, or 46689 polypeptide.

[0017] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 23479, 48120, or 46689 polypeptides or nucleic acids.

[0018] In still another aspect, the invention provides a process for modulating 23479, 48120, or 46689 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 23479, 48120, or 46689 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular proliferation or differentiation.

[0019] In yet another aspect, the invention provides methods for inhibiting the proliferation or inducing the killing, of a 23479, 48120, or 46689-expressing cell, e.g., a hyper-proliferative 23479, 48120, or 46689-expressing cell. The method includes contacting the cell with an agent, e.g., a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 23479, 48120, or 46689 polypeptide or nucleic acid. In a preferred embodiment, the contacting step occurs in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol.

[0020] In one embodiment, the cell is a hyperproliferative cell, e.g., a cell found in a solid tumor, a soft tissue tumor, or a metastatic lesion, e.g., a tumor or metastatic lesion of the lung, brain, ovary or breast.

[0021] In other embodiments, the cell is a neuron or a glial cell, e.g., a cortical or hypothalamic cell. In yet other embodiments, the cell is a cardiovascular cell, e.g., a heart- or blood vessel-associated cell.

[0022] In a preferred embodiment, the agent, e.g., the compound, is an inhibitor of a 23479, 48120, or 46689 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety selected from a cytotoxin, a cytotoxic agent and a radioactive metal ion). In another embodiment, the agent, e.g., the compound, is an inhibitor of a 23479, 48120, or 46689 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule.

[0023] In a preferred embodiment, the agent, e.g., the compound, is administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation.

[0024] In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant cellular proliferation or differentiation of a 23479, 48120, or 46689-expressing cell, in a subject. Preferably, the method includes administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 23479, 48120, or 46689 polypeptide or nucleic acid.

[0025] The disorder can be a cancerous or pre-cancerous condition, e.g., a solid tumor, a soft tissue tumor, or a metastatic lesion, e.g., a tumor or metastatic lesion of the lung, brain, ovary or breast. In other embodiments, the disorder is a neurological or a cardiovascular disorder.

[0026] In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., a cellular proliferative or differentiative disorder, a neurological or a cardiovascular disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with one or more of: chemotherapy, radiation, and/or a compound identified using the methods described herein); and evaluating the expression of a 23479, 48120, or 46689 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 23479, 48120, or 46689 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 23479, 48120, or 46689 nucleic acid or polypeptide expression can be detected by any method described herein.

[0027] In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 23479, 48120, or 46689 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.

[0028] In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein, a cytotoxic agent) and, evaluating the expression of 23479, 48120, or 46689 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 23479, 48120, or 46689 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 23479, 48120, or 46689 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a cancerous tissue or a lung, brain, ovary, or breast tissue.

[0029] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 23479, 48120, or 46689 polypeptide or nucleic acid molecule, including for disease diagnosis.

[0030] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 23479, 48120, or 46689 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 23479, 48120, or 46689 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 23479, 48120, or 46689 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

[0031] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 depicts a hydropathy plot of human 23479 polypeptide. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 23479 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 100 to 110, from about amino acid 295 to 310, and from about amino acid 920 to 930, of SEQ ID NO: 2; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence from about amino acid 275 to 290, from about amino acid 530 to 550, and from about amino acid 640 to 650, of SEQ ID NO: 2.

[0033]FIG. 2A depicts an alignment of the first ubiquitin carboxyl-terminal hydrolase domain (UCH-1) of human 23479 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 10), while the lower amino acid sequence corresponds to amino acids 296-327 of SEQ ID NO: 2.

[0034]FIG. 2B depicts an alignment of the second ubiquitin carboxyl-terminal hydrolase domain (UCH-2) of human 23479 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 11), while the lower amino acid sequence corresponds to amino acids 546-640 of SEQ ID NO: 2.

[0035]FIG. 3 depicts a hydropathy plot of human 48120 polypeptide. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 48120 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 1040 to 1055 of SEQ ID NO: 5; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence from about amino acid 120 to 155, from about amino acid 680 to 700, and from about amino acid 770 to 800, of SEQ ID NO: 5.

[0036]FIG. 4A depicts an alignment of the first ubiquitin carboxyl-terminal hydrolase domain (UCH-1) of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 10), while the lower amino acid sequence corresponds to amino acids 162 to 193 of SEQ ID NO: 5.

[0037]FIG. 4B depicts an alignment of the second ubiquitin carboxyl-terminal hydrolase domain (UCH-2) of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 1), while the lower amino acid sequence corresponds to amino acids 580 to 649 of SEQ ID NO: 5.

[0038]FIG. 4C depicts an alignment of the ubiquitin associated (UBA) domain of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 12), while the lower amino acid sequence corresponds to amino acids 20 to 61 of SEQ ID NO: 5.

[0039]FIG. 4D depicts an alignment of the ubiquitin interaction motif (UIM) domain of human 48120 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 13), while the lower amino acid sequence corresponds to amino acids 96 to 113 of SEQ ID NO: 5.

[0040]FIG. 5 depicts a hydropathy plot of human 46689 polypeptide. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 46689 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 1 to 23, from about 133 to 145, and from about 150 to 168 of SEQ ID NO: 8; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 34 to 51, from about 333 to 347, and from about 438 to 449 of SEQ ID NO: 8.

[0041]FIG. 6 depicts an alignment of the α/β hydrolase domain of human 46689 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 14), while the lower amino acid sequence corresponds to about amino acid residues 186 to 419 of SEQ ID NO: 8.

[0042]FIG. 7 depicts a BLAST alignment of the α/β hydrolase domain of human 46689 with a consensus amino acid sequence derived from a ProDom family PD007763 (Release 2001.1; http://www.toulouse.inra.fr/prodom.html). The lower sequence is the consensus amino acid sequence (SEQ ID NO: 15), while the upper amino acid sequence corresponds to the α/β hydrolase domain of human 46689 along with some flanking sequence, about amino acid residues 97 to 424 of SEQ ID NO: 8.

DETAILED DESCRIPTION

[0043] Human 23479

[0044] The human 23479 sequence (see SEQ ID NO: 1, as recited in Example 1), which is approximately 3494 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2805 nucleotides, including the termination codon. The coding sequence encodes a 934 amino acid protein (see SEQ ID NO: 2, as recited in Example 1).

[0045] Human 23479 contains the following regions or structural features:

[0046] a ubiquitin carboxyl-terminal hydrolase-1 (UCH-1) domain (FIG. 2A; PFAM Accession PF00442) located at about amino acid residues 296-327 of SEQ ID NO: 2;

[0047] a ubiquitin carboxyl-terminal hydrolase-2 (UCH-2) domain (FIG. 2B; PFAM Accession PF00443) located at about amino acid residues 546-640 of SEQ ID NO: 2;

[0048] two predicted N-glycosylation sites (PS00001) located at about amino acid residues 94-97 and 739-742 of SEQ ID NO: 2;

[0049] one predicted cAMP and cGMP-dependent protein kinase phosphorylation site (PS00004) located at about amino acid residues 60-63 of SEQ ID NO: 2;

[0050] twelve predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 228-230, 241-243, 326-328, 402-404, 432-434, 451-453, 490-492, 529-531, 611-613, 619-621, 706-708, and 932-934 of SEQ ID NO: 2;

[0051] sixteen predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 23-26, 48-51, 85-88, 156-159, 228-231, 338-341, 390-393, 426-429, 446-449, 451-454, 617-620, 695-698, 808-811, 890-893, 905-908, and 930-933 of SEQ ID NO: 2;

[0052] three predicted tyrosine kinase phosphorylation sites (PS00007) located at about amino acid residues 22-30, 543-550, and 674-681 of SEQ ID NO: 2;

[0053] seven predicted N-myristoylation sites (PS00008) located at about amino acid residues 86-91, 256-261, 408-413, 560-565, 607-612, 798-803, and 814-819 of SEQ ID NO: 2;

[0054] one predicted amidation site (PS00009) located at about amino acid residues 467-470 of SEQ ID NO: 2;

[0055] one predicted ubiquitin carboxyl-terminal hydrolase family 2 signature 2 (PS00973) located at about amino acid residues 550-567 of SEQ ID NO: 2;

[0056] one peroxisomal targeting signal located at about amino acid residues 785-793 of SEQ ID NO: 2; and

[0057] one predicted coiled coil domain located at about amino acid residues 884-911 of SEQ ID NO: 2.

[0058] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/ software/packages/pfam/pfam.html.

[0059] A plasmid containing the nucleotide sequence encoding human 23479 (clone “Fbh23479FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______ .This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0060] Human 48120

[0061] The human 48120 sequence (see SEQ ID NO: 4, as recited in Example 1), which is approximately 4873 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 3420 nucleotides, including the termination codon. The coding sequence encodes a 1139 amino acid protein (see SEQ ID NO: 5, as recited in Example 1).

[0062] Human 48120 contains the following regions or structural features:

[0063] a ubiquitin carboxyl-terminal hydrolase-1 (UCH-1) domain (FIG. 4A; PFAM Accession PF00442) located at about amino acid residues 162-193 of SEQ ID NO: 5;

[0064] a ubiquitin carboxyl-terminal hydrolase-2 (UCH-2) domain (FIG. 4B; PFAM Accession PF00443) located at about amino acid residues 580-649 of SEQ ID NO: 5;

[0065] a ubiquitin associated (UBA) domain (FIG. 4C; PFAM Accession PF00627) located at about amino acid residues 20-61 of SEQ ID NO: 5;

[0066] a ubiquitin interaction motif (UIM) domain (FIG. 4D; PFAM Accession PF02809) located at about amino acid residues 96-113 of SEQ ID NO: 5;

[0067] six predicted N-glycosylation sites (PS00001) located at about amino acid residues 282-285, 310-313, 373-376, 639-642, 711-714, and 916-919 of SEQ ID NO: 5;

[0068] one predicted cAMP and cGMP-dependent protein kinase phosphorylation site (PS00004) located at about amino acid residues 958-961 of SEQ ID NO: 5;

[0069] fifteen predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 113-115, 134-136, 137-139, 207-209, 228-230, 260-262, 279-281, 347-349, 453-455, 484-486, 517-519, 700-702, 753-755, 1110-1112, and 1137-1139 of SEQ ID NO: 5;

[0070] thirty-three predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 47-50, 76-79, 109-112, 130-133, 205-208, 248-251, 368-371, 479-482, 484-487, 489-492, 494-497, 503-506, 520-523, 532-535, 550-553, 620-623, 624-627, 662-665 668-671, 713-716, 719-722, 760-763, 808-811, 822-825, 881-884, 907-910, 918-921, 966-969 1024-1027, 1028-1031, 1033-1036, 1058-1061, 1115-1118of SEQID NO: 5;

[0071] one predicted tyrosine kinase phosphorylation site (PS00007) located at about amino acid residues 975-982 of SEQ ID NO: 5;

[0072] thirteen predicted N-myristoylation sites (PS00008) located at about amino acid residues 12-17, 80-85, 244-249, 294-299, 300-305, 433-438, 594-599, 635-640, 761-766, 839-844, 855-860, 1001-1006, and 1077-1082 of SEQID NO: 5;

[0073] one predicted carbamoyl-phosphate synthase subdomain signature 2 (PS00867) located at about amino acid residues 1015-1022 of SEQ ID NO: 5;

[0074] one predicted ubiquitin carboxyl-terminal hydrolase family 2 signature 2 (PS00973) located at about amino acid residues 584-601 of SEQ ID NO: 5; and

[0075] one predicted coiled coil domain located at about amino acid residues 399-431 of SEQ ID NO: 5.

[0076] A plasmid containing the nucleotide sequence encoding human 48120 (clone “Fbh48120FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______ . This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0077] Human 46689

[0078] The human 46689 sequence (see SEQ ID NO: 1, as recited in Example 1), which is approximately 2082 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1407 nucleotides, including the termination codon. The coding sequence encodes a 468 amino acid protein (see SEQ ID NO: 2, as recited in Example 1). The human 46689 protein of SEQ ID NO: 2 and FIG. 2 includes an amino-terminal hydrophobic amino acid sequence, consistent with a signal sequence, of about 26 amino acid residues (from amino acid 1 to about amino acid 26 of SEQ ID NO: 2), which upon cleavage results in the production of a mature protein form. This mature protein form is approximately 442 amino acid residues in length (from about amino acid residues 27 to 468 of SEQ ID NO: 2).

[0079] Human 46689 contains the following regions or other structural features:

[0080] an α/β hydrolase domain (PFAM Accession Number PF00561) located at about amino acid residues 186 to 419 of SEQ ID NO: 2;

[0081] a predicted catalytic acid residue, located at about amino acid residue 360 of SEQ ID NO: 2;

[0082] a predicted catalytic histidine residue, located about amino acid residue 391 of SEQ ID NO: 2

[0083] a predicted signal peptide located at about amino acid residues 1 to 26 of SEQ ID NO: 2;

[0084] one predicted transmembrane domain located at about amino acid residues 150 to 167 of SEQ ID NO: 2;

[0085] four predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 203 to 205, 305 to 307, 313 to 315, and 411 to 413 of SEQ ID NO: 2;

[0086] four predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 297 to 300, 313 to 316, 357 to 360, and 454 to 457 of SEQ ID NO: 2;

[0087] one predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acid residues 148 to 151 of SEQ ID NO: 2;

[0088] two predicted amidation sites (PS00009) located at about amino acid residues 146 to 149, and 437 to 440 of SEQ ID NO: 2: and

[0089] five predicted N-myristylation sites (PS00008) located at about amino acid residues 5 to 10, 52 to 57, 154 to 159, 237 to 242, and 389 to 394 of SEQ ID NO: 2.

[0090] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[0091] A plasmid containing the nucleotide sequence encoding human 46689 (clone “Fbh46689FL”) was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on and assigned Accession Number ______ . This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112. TABLE 1 Summary of Sequence Information for 23479, 48120, and 46689 ATCC Accession Gene cDNA ORF Polypeptide Figure Number 23479 SEQ ID NO:1 SEQ ID NO:3 SEQ ID NO:2 FIG. 1, 2A-2B 48120 SEQ ID NO:4 SEQ ID NO:6 SEQ ID NO:5 FIG. 3, 4A-4D 46689 SEQ ID NO:7 SEQ ID NO:9 SEQ ID NO:8 FIG. 5, 6, 7

[0092] 23479 and 48120 Polypeptides

[0093] The 23479 and 48120 proteins contain a significant number of structural characteristics in common with members of the ubiquitin carboxyl-terminal hydrolase family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also ha,e common functional characteristics.

[0094] Members of the ubiquitin carboxyl-terminal hydrolase family of proteins are characterized by a “ubiquitin carboxyl-terminal hydrolase domain.” The term “ubiquitin carboxyl-terminal hydrolase domain” refers to an amino acid sequence that participates in the removal of one or more ubiquitin molecules from a protein that has one or more molecules of ubiquitin attached to it. The term also includes amino acid sequences that cleave conjugated forms of ubiquitin (e.g., in a head to tail orientation linked via a peptide bond) whether or not the ubiquitin conjugate is attached to a protein. For example, a ubiquitin-ubiquitin conjugate (dimer) could be cleaved into monomers, a tri-ubiquitin conjugate could be cleaved into three monomers, or a dimer and a single monomer. In either of these particular examples, the monomer or dimer could remain attached to or be cleaved from the ubiquitinated protein.

[0095] Ubiquitin carboxyl-terminal hydrolases typically contain two conserved regions, a UCH-1 domain and a UCH-2 domain, each of which is thought to participate in the catalytic mechanism. The conserved signature patterns of UCH-1 and UCH-2 are respectively as follows: (1) G-[LIVMFY]-x(1,3)-[AGC]-[NASM]-x-C-[FYW]-[LIVMFC]-[NST]-[SACV]-x-[LIVMS]-Q; and (2) Y-x-L-x-[SAG]-[LIVMFT]-x (2)-H-x-G-x(4,5)-G-H-Y (SEQ ID NO: 16). 23479 and 48120 proteins preferably contain one or more sequences that conform to this signature pattern.

[0096] A 23479 or 48120 polypeptide can include a “UCH-1 domain” or regions homologous with a “UCH-1 domain.”

[0097] As used herein, the term “UCH-1 domain” includes an amino acid sequence of about 10 to 100 amino acid residues in length and having a bit score for the alignment of the sequence to the UCH-1 domain (HMM) of at least 25. 23479 or 48120 proteins preferably contain sequences that conform to UCH-1 signature pattern described above. Preferably, a 23479 protein contains the sequence GLTNLGATCYLASTIQ (SEQ ID NO: 17). Preferably, a 48120 protein contains the sequence GLKNVGNTCWFSAVIQ (SEQ ID NO: 18). Preferably, a UCH-1 domain includes at least about 20 to 50 amino acids, more preferably about 25 to 40 amino acid residues, or about 30 to 35 amino acids and has a bit score for the alignment of the sequence to the UCH-1 domain (HMM) of at least 45 or greater. The UCH-1 domain has been assigned the PFAM Accession PF00442 (http;//genome.wustl.edu/Pfam/html). Alignments of the UCH-1 domains of human 23479 and 48120 with consensus amino acid sequences derived from hidden Markov models are depicted in FIG. 2A (23479; amino acids 296 to 327 of SEQ ID NO: 2) and FIG. 4A (48120; amino acids 162 to 193 of SEQ ID NO: 5).

[0098] In a preferred embodiment 23479 or 48120 polypeptide or protein has a “UCH-1 domain” or a region which includes at least about 20 to 50 more preferably about 25 to 40 or 30 to 35 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “UCH-1 domain,” e.g., a UCH-1 domain of human 23479 or 48120, e.g., residues 296 to 327 of SEQ ID NO: 2 or residues 162 to 193 of SEQ ID NO: 5.

[0099] A 23479 or 48120 polypeptide can further include a “UCH-2 domain” or regions homologous with a “UCH-2 domain.”

[0100] As used herein, the term “UCH-2 domain” includes an amino acid sequence of about 10 to 150 amino acid residues in length and having a bit score for the alignment of the sequence to the UCH-2 domain (HMM) of at least 50. 23479 or 48120 proteins preferably contain sequences that conform to UCH-2 signature pattern described above. Preferably, a 23479 protein contains the sequence YDLIGVTVHTGTADGGHY (SEQ ID NO: 19). Preferably, a 48120 protein contains the sequence YRLHAVLVHEGQANAGHY (SEQ ID NO: 20). Preferably, a UCH-2 domain includes at least about 30 to 125 amino acids, more preferably about 50 to 110 amino acid residues, or about 60 to 100 amino acids and has a bit score for the alignment of the sequence to the UCH-2 domain (HMM) of at least 75 or greater. The UCH-2 domain has been assigned the PFAM Accession PF00443 (http;//genome.wustl.edu/Pfam/.html). Alignments of the UCH-2 domains of human 23479 and 48120 with consensus amino acid sequences derived from hidden Markov models are depicted in FIG. 2B (23479; amino acids 546 to 640 of SEQ ID NO: 2) and FIG. 4B (48120; amino acids 580 to 649 of SEQ ID NO: 5).

[0101] In a preferred embodiment 23479 or 48120 polypeptide or protein has a “UCH-2 domain” or a region which includes at least about 30 to 125 more preferably about 50 to 110 or 60 to 100 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “UCH-2 domain,” e.g., a UCH-2 domain of human 23479 or 48120, e.g., residues 546 to 640 of SEQ ID NO: 2 or residues 580 to 649 of SEQ ID NO: 5.

[0102] A 48120 polypeptide can also include a “UBA domain” or regions homologous with a “UBA domain.” A “UBA domain” refers to a commonly occurring amino acid sequence found in several proteins having connections to ubiquitin and the ubiquitination pathway. The structure of the UBA domain consists of a compact three helix bundle.

[0103] As used herein, the term “UBA domain” includes an amino acid sequence of about 10 to 100 amino acid residues in length and having a bit score for the alignment of the sequence to the UBA domain (HMM) of at least 5. Preferably, a UBA domain includes at least about 20 to 80 amino acids, more preferably about 25 to 60 amino acid residues, or about 35 to 45 amino acids and has a bit score for the alignment of the sequence to the UBA domain (HMM) of at least 8 or greater. The UBA domain (HMM) has been assigned the PFAM Accession Number PF00627 (http://genome.wustl.edu/Pfam/html). An alignment of the UBA domain (amino acids 20-61 of SEQ ID NO: 5) of human 48120 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 4C.

[0104] In a preferred embodiment a 48120 polypeptide or protein has a “UBA domain” or a region which includes at least about 20 to 80 more preferably about 25 to 60 or 35 to 45 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “UBA domain,” e.g., a UBA domain of human 48120, e.g., residues 20 to 61 of SEQ ID NO: 5.

[0105] A 48120 polypeptide can also include a “ubiquitin interaction motif (UIM) domain” or regions homologous with a “UIM domain.”

[0106] As used herein, the term “UIM domain” includes an amino acid sequence of about 10 to 50 amino acid residues in length and having a bit score for the alignment of the sequence to the UBA domain (HMM) of at least 5. Preferably, a UIM domain includes at least about 10 to 40 amino acids, more preferably about 10 to 30 amino acid residues, or about 15 to 20 amino acids and has a bit score for the alignment of the sequence to the UIM domain (HMM) of at least 10 or greater. The UIM domain (HMM) has been assigned the PFAM Accession Number PF02809 (http://genome.wustl.edu/Pfam/html). An alignment of the UIM domain (amino acids 96-113 of SEQ ID NO: 5) of human 48120 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 4D.

[0107] In a preferred embodiment a 48120 polypeptide or protein has a “UIM domain” or a region which includes at least about 10 to 40 more preferably about 10 to 30 or 15 to 20 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “UIM domain,” e.g., a UIM domain of human 48120, e.g., residues 96 to 113 of SEQ ID NO: 5.

[0108] To identify the presence of a UCH-1, UCH-2, UBA, or UIM domain in a 23479 or 48120 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al.(1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994)J. Mol. Biol. 235:1501-1531; and Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

[0109] A 23479 or 48120 family member can include a UCH-1 domain and a UCH-2 domain. A 48120 family member can also include a UBA domain. A 48120 family member can further include a UIM domain.

[0110] A 23479 family member can also include at least one and preferably two N-glycosylation sites (PS00001); at least one cAMP and cGMP-dependent protein kinase phosphorylation site (PS00004); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, and preferably 12 protein kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, and preferably 16 casein kinase II phosphorylation sites (PS00006); at least one, two, and preferably three tyrosine kinase phosphorylation sites (PS00007); at least one, two, three, four, five, six, and preferably seven N-myristoylation sites (PS00008); at least one amidation site (PS00009); at least one ubiquitin carboxyl-terminal hydrolase family 2 signature 2 (PS00973); at least one peroxisomal targeting signal; and at least one coiled coil domain.

[0111] A 48120 family member can also include at least one, two, three, four, five, and preferably six N-glycosylation sites (PS00001); at least one cAMP and cGMP-dependent protein kinase phosphorylation site (PS00004); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, and preferably 15 protein kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and preferably 33 casein kinase II phosphorylation sites (PS00006); at least one tyrosine kinase phosphorylation site (PS00007); at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, and preferably 13 N-myristoylation sites (PS00008); at least one carbamoyl-phosphate synthase subdomain signature 2 (PS00867); at least one ubiquitin carboxyl-terminal hydrolase family 2 signature 2 (PS00973); and at least one coiled coil domain.

[0112] As the 23479 or 48120 polypeptides of the invention may modulate 23479 or 48120-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 23479 or 48120-mediated or related disorders, as described below.

[0113] As used herein, a “23479 or 48120 activity”, “biological activity of 23479 or 48120” or “functional activity of 23479 or 48120”, refers to an activity exerted by a 23479 or 48120 protein, polypeptide or nucleic acid molecule. For example, a 23479 or 48120 activity can be an activity exerted by 23479 or 48120 in a physiological milieu on, e.g., a 23479 or 48120-responsive cell or on a 23479 or 48120 substrate, e.g., a protein substrate. A 23479 or 48120 activity can be determined in vivo or in vitro. In one embodiment, a 23479 or 48120 activity is a direct activity, such as an association with a 23479 or 48120 target molecule. A “target molecule” or “binding partner” is a molecule with which a 23479 or 48120 protein binds or interacts in nature, e.g., a complex of ubiquitin and a protein targeted for degradation.

[0114] A 23479 or 48120 activity can also be an indirect activity, e.g., an activity mediated by a protein that is a target for de-ubiquitination by 23479 or 48120. The features of the 23479 or 48120 molecules of the present invention can provide similar biological activities as ubiquitin carboxyl-terminal hydrolase family members. For example, the 23479 or 48120 proteins of the present invention can have one or more of the following activities: 1) modulation of de-ubiquitination of a substrate, e.g., a ubiquitinated protein targeted for degradation; 2) participation in the processing of poly-ubiquitin precursors; 3) modulation of cellular proliferation and/or differentiation; 4) modulation of apoptosis; 5) modulation of transcription and/or cell-cycle progression; 6) modulation of signal-transduction; 7) modulation of antigen processing; 8) modulation of cell-cell adhesion; 9) modulation of receptor-mediated endocytosis; 10) modulation of organelle biogenesis and development; 11) participation in neuropathological conditions; and 12) participation in oncogenesis.

[0115] Based on the above-described sequence similarities, the 23479 or 48120 molecules of the present invention are predicted to have similar biological activities as ubiquitin carboxyl-terminal hydrolase family members. Ubiquitin carboxyl-terminal hydrolase domains regulate the de-ubiquitination of a substrate, e.g., a protein targeted for degradation. Thus, 23479 or 48120 molecules can act as novel diagnostic targets and therapeutic agents for controlling, e.g., ubiquitination related disorders. 23479 or 48120 molecules of the invention may be useful, for example, in inducing the de-ubiquitination of ubiquitinated proteins. These proteins can therefore modulate protein degradation and the recycling of ubiquitin, as well as participate in cell signaling pathways in which ubiquitination or de-ubiquitination of a protein can alter or modify the activity of the protein. Thus, 23479 or 48120 molecules may act as novel therapeutic agents for controlling disorders associated with excessive or insufficient ubiquitination (e.g., protein degradation), and as diagnostic markers useful for indicating the presence or predisposition towards developing such disorders, or monitoring the progression or regression of a disorder.

[0116] Ubiquitination has been implicated in regulating numerous cellular processes including, for example, proliferation, differentiation, apoptosis (programmed cell death), transcription, signal-transduction, cell-cycle progression, receptor-mediated endocytosis, organelle biogenesis and others. The presence of abnormal amounts of ubiquitinated proteins in neuropathological conditions such as Alzheimer's and Pick's disease indicates that ubiquitination plays a role in various physiological disorders. Oncogenes (e.g., v-jun and v-fos) are often found to be resistant to ubiquitination in comparison to their normal cell counterparts, suggesting that a failure to degrade oncogene protein products accounts for some of their cell transformation capability.

[0117] As the 23479 and 48120 molecules of the invention are expressed in coronary and endothelial tissues, brain tissues, erythroid cells, and lung tumors (See Example 2), they can act as novel diagnostic targets and therapeutic agents for controlling disorders associated with abnormal de-ubiquitination activity and disorders associated with abnormal protein degradation in such tissues. Thus, examples of disorders that can be treated and/or diagnosed with the molecules of the invention include cellular proliferative and/or differentiative disorders (e.g., in the lung), cardiovascular disorders, brain disorders, and hematopoietic disorders.

[0118] 46689 polypeptides

[0119] The 46689 protein contains a significant number of structural characteristics in common with members of the α/β hydrolase family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[0120] The α/β hydrolase family of proteins is characterized by a common fold. In its most typical form, the fold consists of an eight stranded β-sheet surrounded by α-helices which further includes conserved catalytic residues. This enzyme family includes lipases, esterases, and proteases. Despite the variety of reactions this family is capable of mediating, the chemistry of these reactions is generally similar. Three positions in particular form a catalytic triad, contributing nucleophilic, acidic, and histidine residues. The side chains of these residues are required for nucleophilic attack on one of the atoms (typically a carbon atom) involved in the chemical bond that is to be cleaved, and thus they function during one of the most critical steps in the catalytic process. The nucleophilic residue is typically serine, although a cysteine or an aspartate residue substitutes for serine in some members of the family. The nucleophilic residue is located in a motif that has been termed the “nucleophile elbow”, a loop that makes a sharp turn following the fifth β-strand of the canonical α/β hydrolase fold. Due to the constrained arrangement of the residues that form the nucleophile elbow, the sequence of the nucleophile elbow typically includes two or three glycine residues. Following the nucleophilic residue is the acidic residue, which is located on a loop following the seventh β-strand. This acidic residue can be either an aspartic acid or a glutamic acid residue. Finally, the third residue of the catalytic triad, the histidine residue, is absolutely conserved and is located in a loop that follows the eighth β-strand of the canonical α/β hydrolase domain. Importantly, the relative order of these three catalytic residues within a given α/β hydrolase peptide sequence, nuclophile-acid-histidine, is conserved in all members of the α/β hydrolase family. Another conserved feature of the α/β hydrolase fold is an oxyanion hole, located near the end of the third β-strand, which is believed to stabilize potential covalent intermediates formed during the nucleophilic attack step in the catalytic process. The covalent intermediate then proceeds to product by general base catalysis. A detailed description of the α/β hydrolase fold can be found in Ollis et al. (1992), Protein Eng 5(3):197-211, and Nardini and Dijkstra (1999), Curr Opin Struct Biol 9(6):732-7, the contents of which are incorporated herein by reference.

[0121] A 46689 polypeptide can include an “α/β hydrolase domain” or regions homologous with an “α/β hydrolase domain”.

[0122] As used herein, the term “α/β hydrolase domain” includes an amino acid sequence of about 100 to 350 amino acid residues in length which contains a conserved catalytic triad consisting of a nucleophilic amino acid residue, an acidic amino acid residue, and a histidine residue. Preferably, an α/β hydrolase domain includes at least about 150 to 300 amino acids, more preferably about 175 to 250 amino acid residues, or about 200 to 250 amino acids. Based on sequence alignments, the presence and extent of an α/β hydrolase domain in a test protein sequence can be determined. One description of an α/β hydrolase domain (HMM) has been assigned the PFAM Accession Number PF00561 (http://pfam.wustl.edu). An alignment of human 46689 (about amino acids 186 to 419 of SEQ ID NO: 8) with the PFAM α/β hydrolase domain consensus amino acid sequence (SEQ ID NO: 14) derived from a hidden Markov model is depicted in FIG. 6. The alignment demonstrates the presence of a catalytic acid residue, located at about amino acid 360 of SEQ ID NO: 8, and a catalytic histidine residue, located about amino acid residue 391 of SEQ ID NO: 8. The alignment also suggests that the serine residue located at about amino acid residue 238 of SEQ ID NO: 8 may be the catalytic nucleophile residue of human 46689.

[0123] A consensus sequence for α/β hydrolase domain-containing protein families is also provided, e.g., by ProDom family PD007763 (ProDomain Release 2001.1; http://www.toulouse.inra.fr/prodom.html). An alignment of a large portion of human 46689 (about amino acid residues 97 to 424 of SEQ ID NO: 8) with the consensus amino acid sequence of an alp hydrolase-containing family of proteins (SEQ ID NO: 15) derived from recursive PSI-BLAST searches, is depicted in FIG. 7. This alignment also reveals the presence of the conserved catalytic acid and catalytic histidine residues of human 46689 polypeptides.

[0124] In a preferred embodiment, a 46689 polypeptide or protein has an “α/β hydrolase domain” or a region which includes a conserved catalytic triad consisting of a nucleophilic residue, an acid residue, and a histidine residue, wherein the nucleophilic residue is separated from the catalytic acid residue by about 110 to 145 amino acid residues, more preferably about 115 to 130 amino acid residues, or about 122 amino acid residues.

[0125] In another preferred embodiment, a 46689 polypeptide or protein has an “α/β hydrolase domain” or a region which includes a conserved catalytic triad consisting of a nucleophilic residue, an acid residue, and a histidine residue, wherein the catalytic acid residue is separated from the catalytic histidine residue by about 15 to 40 amino acid residues, more preferably about 25 to 35 amino acid residues, or about 31 amino acid residues.

[0126] In yet another preferred embodiment, a 46689 polypeptide or protein has an “α/β hydrolase domain” or a region which includes at least about 100 to 350, more preferably about 175 to 300, or 200 to 250 amino acid residues and has at least about 70% 80% 90% 95%, 98% 99%, or 100% homology with an “α/β hydrolase domain,” e.g., the α/β hydrolase domain of human 46689 (e.g., residues 186 to 419 of SEQ ID NO: 8).

[0127] To identify the presence of an “α/β hydrolase” domain in a 46689 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the PFAM database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the PFAM database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of an “α/β hydrolase” domain in the amino acid sequence of human 46689 at about residues 186 to 419 of SEQ ID NO: 2 (see FIG. 6).

[0128] A 46689 molecule can further include an amino acid sequence homologous to “ProDom PD007763 domain.” Members of the family of proteins containing this domain are uncharacterized proteins that share conserved regions, including a hydrolase signature. Yeast protein YMR210W and human protein PHPS1-2 are members of this family.

[0129] As used herein, the term “ProDom PD007763 domain” includes an amino acid sequence of about 250 to 450 amino acid residues in length having a bit score for the alignment of the sequence with ProDom PD007763 of at least 50. Preferably, a ProDom PD007763 domain includes at least 250 to 450 amino acids, more preferably about 275 to 400 amino acid residues, or about 300 to 350 amino acids, and has a bit score for the alignment of the sequence with ProDom PD007763 of at least 75, 85, preferably 95 or more. An alignment of the ProDom PD007763 domain of human 46689 (amino acids 97 to 424 of SEQ ID NO: 8) with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 7.

[0130] In a preferred embodiment, a 46689 polypeptide or protein has a “ProDom PD007763 domain” or a region which includes at least about 250 to 450, more preferably about 275 to 400, or 300 to 350 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “ProDom PD007763 domain”, e.g., the ProDom PD007763 domain of human 46689 (e.g., residues 97 to 424 of SEQ ID NO: 8).

[0131] To identify the presence of a “ProDom PD007763 domain” in a 46689 protein sequence, and make the determination that a polypeptide or protein of interest contains such a domain, the amino acid sequence of the protein can be searched against the ProDom database (Corpet et al. (1999), Nucl. Acids Res. 27:263-267) The ProDom protein domain database consists of an automatic compilation of homologous domains. Current versions of ProDom are built using recursive PSI-BLAST searches (Altschul SF et al. (1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers and Chemistry 23:333-340.) of the SWISS-PROT 38 and TREMBL protein databases. The database automatically generates a consensus sequence for each domain. A BLAST search was performed against the ProDom database resulting in the identification of an “α/β hydrolase” domain within the amino acid sequence of human 46689 that includes residues 97 to 424 of SEQ ID NO: 8 (FIG. 7).

[0132] A 46689 protein can further include at least one transmembrane domain. As used herein, the term “transmembrane domain” includes an amino acid sequence of at least about 10 amino acid residues in length that spans the plasma membrane. More preferably, a transmembrane domain includes about at least 15 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al., (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. Amino acid residues 150 to 167 of the 46689 protein (SEQ ID NO: 8) are predicted to be a transmembrane domain (see FIG. 5). Accordingly, 56294 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with at least one transmembrane domain of human 46689 are within the scope of the invention.

[0133] In a preferred embodiment, 46689 protein has a “transmembrane domain” or a region which includes at least about 10, more preferably at least about 15 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “transmembrane domain,” e.g., the transmembrane domain of 46689 protein (e.g., residues 150 to 167 of SEQ ID NO: 8).

[0134] A 46689 protein can further include a signal sequence. As used herein, a “signal peptide” or “signal sequence” refers to a peptide of about 15 to 60, preferably about 20 to 40, more preferably, 27 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains a majority of hydrophobic amino acid residues. For example, a signal sequence contains at least about 15 to 60, preferably about 20 to 40, more preferably, 27 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g., alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a “signal sequence”, also referred to in the art as a “signal peptide”, serves to direct a protein containing such a sequence to a lipid bilayer. For example, in one embodiment, a 46689 protein contains a signal sequence of about 26 amino acids. The “signal sequence” is cleaved during processing of the mature protein. The mature 46689 protein corresponds to amino acids 27 to 468 of SEQ ID NO: 8.

[0135] A 46689 family member can include at least one α/β hydrolase domain and/or at least one ProDom PD007763 domain. Furthermore, a 46689 family member can include at least one catalytic acid residue; at least one catalytic histidine residue; at least one transmembrane domain; at least one signal peptide; at least one, two, three, preferably four predicted protein kinase C phosphorylation sites (PS00005); at least one, two, three, preferably four predicted casein kinase II phosphorylation sites (PS00006); at least one predicted cAMP- and cGMP-dependent protein kinase phosphorylation site (PS00004); at least one, preferably two predicted amidation sites (PS00009); and at least one, two, three, four, preferably five predicted N-myristylation sites (PS00008).

[0136] As the 46689 polypeptides of the invention may modulate 46689-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 46689-mediated or related disorders, as described below.

[0137] As used herein, a “46689 activity”, “biological activity of 46689” or “functional activity of 46689”, refers to an activity exerted by a 46689 protein, polypeptide or nucleic acid molecule. For example, a 46689 activity can be an activity exerted by 46689 in a physiological milieu on, e.g., a 46689-responsive cell or on a 46689 substrate, e.g., a protein, lipid, or small molecule substrate. A 46689 activity can be determined in vivo or in vitro. In one embodiment, a 46689 activity is a direct activity, such as an association with a 46689 target molecule. A “target molecule” or “binding partner” is a molecule with which a 46689 protein binds or interacts in nature. In an exemplary embodiment, 46689 hydrolyzes a substrate, e.g., a protein, lipid, or small molecule (e.g., metabolite, signaling molecule, toxin, or carcinogen) substrate.

[0138] A 46689 activity can also be an indirect activity, e.g., modulation of a cellular signaling activity mediated by a 46689 substrate or product. The features of the 46689 molecules of the present invention can provide similar biological activities as α/β hydrolase family members. For example, the 46689 proteins of the present invention can have one or more of the following activities: (1) hydrolysis of lipid substrates; (2) hydrolysis of cholesterol; (3) hydrolysis of epoxides and other toxic chemicals; (4) hydrolysis of acetylcholine and other neurotransmitters; (5) protease activity; (6) hydrolysis of carboxylesters; or (7) thioesterase activity. As a result, the 46689 protein may have a critical function in one or more of the following physiological processes: (1) metabolite regulation and degradation; (2) drug metabolism; (3) toxin or carcinogen removal and neutralization; (4) toxin or carcinogen production; (4) cellular proliferation or differentiation; and (5) neuronal function.

[0139] The 46689 polypeptide may be involved in disorders of metabolic imbalance, including obesity, anorexia nervosa, cachexia, lipid disorders, cholesterol imbalance, and diabetes. For example, many α/β hydrolase family members have lipase activity and cholesterol esterase activity and the human hormone sensitive lipase is responsible for metabolizing fat stored in adipocytes. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, α1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease).

[0140] The 46689 polypeptide may be involved in disorders of toxin (e.g., carcinogen) metabolism and/or removal. For example, disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol. 46689 molecules may also have a critical role in removing xenobiotic epoxides and other toxins from the body. Furthermore, it may contribute to the metabolism of drugs and other pharmaceuticals. The study of polymorphisms in the 46689 gene should provide a useful resource for pharmacogenomic (see below) analysis of drug responses. Additionally, variations in 46689 may contribute to population differences in sensitivity to environmental toxins.

[0141] In addition, as the 46689 molecules are expressed in bone marrow tissue, lung tissue, thymus tissue, glial cells, brain tissue, and kidney tissue, as well as in lung, brain, ovary, and breast tumors, they can act as novel diagnostic targets and therapeutic agents for controlling disorders associated with abnormal cellular metabolism in those tissues.

[0142] Thus, examples of disorders that can be treated and/or diagnosed with the molecules of the invention include cellular proliferative and/or differentiative disorders (e.g., in the lung, brain, ovary, or breast), hematopoietic disorders, neural disorders (e.g., brain disorders), liver disorders, and cardiovascular disorders.

[0143] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[0144] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[0145] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0146] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0147] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0148] Examples of cellular proliferative and/or differentiative disorders of the colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

[0149] Examples of cellular proliferative and/or differentiative disorders of the liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.

[0150] Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

[0151] Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

[0152] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[0153] As used herein, heart disorders, or “cardiovascular disease” or a “cardiovascular disorder” includes a disease or disorder which affects the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. A cardiovascular disorder includes, but is not limited to disorders such as arteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, valvular disease, including but not limited to, valvular degeneration caused by calcification, rheumatic heart disease, endocarditis, or complications of artificial valves; atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, pericardial disease, including but not limited to, pericardial effusion and pericarditis; cardiomyopathies, e.g., dilated cardiomyopathy or idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, ischemic disease, arrhythmia, sudden cardiac death, and cardiovascular developmental disorders (e.g., arteriovenous malformations, arteriovenous fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome, causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrial septal defects, atrioventricular canal, coarctation of the aorta, ebsteins anomaly, hypoplastic left heart syndrome, interruption of the aortic arch, mitral valve prolapse, ductus arteriosus, patent foramen ovale, partial anomalous pulmonary venous return, pulmonary atresia with ventricular septal defect, pulmonary atresia without ventricular septal defect, persistance of the fetal circulation, pulmonary valve stenosis, single ventricle, total anomalous pulmonary venous return, transposition of the great vessels, tricuspid atresia, truncus arteriosus, ventricular septal defects). A cardiovasular disease or disorder also can include an endothelial cell disorder.

[0154] As used herein, an “endothelial cell disorder” includes a disorder characterized by aberrant, unregulated, or unwanted endothelial cell activity, e.g., proliferation, migration, angiogenesis, or vascularization; or aberrant expression of cell surface adhesion molecules or genes associated with angiogenesis, e.g., TIE-2, FLT and FLK. Endothelial cell disorders include tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy, endometriosis, Grave's disease, ischemic disease (e.g., atherosclerosis), and chronic inflammatory diseases (e.g., rheumatoid arthritis).

[0155] Examples of hematopoieitic disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

[0156] Disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin B1) deficiency and vitamin B12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.

[0157] Disorders which may be treated or diagnosed by methods described herein include, but are not limited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, A1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

[0158] The 23479, 48120, or 46689 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 thereof are collectively referred to as “polypeptides or proteins of the invention” or “23479, 48120, or 46689 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “23479, 48120, or 46689 nucleic acids.” 23479, 48120, or 46689 molecules refer to 23479, 48120, or 46689 nucleic acids, polypeptides, and antibodies.

[0159] As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0160] The term “isolated nucleic acid molecule” or “purified nucleic acid molecule” includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0161] As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[0162] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9 corresponds to a naturally-occurring nucleic acid molecule.

[0163] As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein.

[0164] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules that include at least an open reading frame encoding a 23479, 48120, or 46689 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 23479, 48120, or 46689 protein or derivative thereof.

[0165] An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. “Substantially free” means that a preparation of 23479, 48120, or 46689 protein is at least 10% pure. In a preferred embodiment, the preparation of 23479, 48120, or 46689 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-23479, 48120, or 46689 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-23479, 48120, or 46689 chemicals. When the 23479, 48120, or 46689 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0166] A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 23479, 48120, or 46689 without abolishing or substantially altering a 23479, 48120, or 46689 activity. Preferably the alteration does not substantially alter the 23479, 48120, or 46689 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of 23479, 48120, or 46689, results in abolishing a 23479, 48120, or 46689 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 23479, 48120, or 46689 are predicted to be particularly unamenable to alteration.

[0167] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 23479, 48120, or 46689 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 23479, 48120, or 46689 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 23479, 48120, or 46689 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0168] As used herein, a “biologically active portion” of a 23479, 48120, or 46689 protein includes a fragment of a 23479, 48120, or 46689 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 23479, 48120, or 46689 molecule and a non-23479, 48120, or 46689 molecule or between a first 23479, 48120, or 46689 molecule and a second 23479, 48120, or 46689 molecule (e.g., a dimerization interaction). Biologically active portions of a 23479, 48120, or 46689 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 23479, 48120, or 46689 protein, e.g., the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, which include less amino acids than the full length 23479, 48120, or 46689 proteins, and exhibit at least one activity of a 23479, 48120, or 46689 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 23479, 48120, or 46689 protein, e.g., hydrolysis of a substrate molecule, e.g., a protein (e.g., a ubiquitinated protein or poly-ubiquitin), lipid, or small molecule (e.g., metabolite, signaling molecule, toxin, or carcinogen) substrate. A biologically active portion of a 23479, 48120, or 46689 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 23479, 48120, or 46689 protein can be used as targets for developing agents which modulate a 23479, 48120, or 46689 mediated activity, e.g., hydrolysis of a substrate molecule, e.g., a protein (e.g., a ubiquitinated protein or poly-ubiquitin), lipid, or small molecule (e.g., metabolite, signaling molecule, toxin, or carcinogen) substrate.

[0169] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

[0170] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0171] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0172] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0173] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0174] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 23479, 48120, or 46689 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 23479, 48120, or 46689 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0175] Particularly preferred 23479, 48120, or 46689 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 are termed substantially identical.

[0176] In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9 are termed substantially identical. “Misexpression or aberrant expression”, as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, translated amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[0177] “Subject,” as used herein, refers to human and non-human animals. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[0178] A “purified preparation of cells”, as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[0179] Various aspects of the invention are described in further detail below.

[0180] Isolated Nucleic Acid Molecules

[0181] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 23479, 48120, or 46689 polypeptide described herein, e.g., a full-length 23479, 48120, or 46689 protein or a fragment thereof, e.g., a biologically active portion of 23479, 48120, or 46689 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide of the invention, 23479, 48120, or 46689 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[0182] In one embodiment, an isolated nucleic acid molecule of the invention includes the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 23479, 48120, or 46689 protein (i.e., “the coding region” of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, as shown in SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9, respectively), as well as 5′ untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7 (e.g., SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9, respectively) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment of a 46689 protein from about amino acid 27 to 468 of SEQ ID NO: 2.

[0183] In another embodiment, an isolated nucleic acid molecule of the invention includes a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule of the invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9, thereby forming a stable duplex.

[0184] In one embodiment, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence which is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9, or a portion, preferably of the same length, of any of these nucleotide sequences.

[0185] 23479 and 48120 Nucleic Acid Fragments

[0186] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6. For example, such a nucleic acid molecule can include a fragment that can be used as a probe or primer or a fragment encoding a portion of a 23479 or 48120 protein, e.g., an immunogenic or biologically active portion of a 23479 or 48120 protein. A fragment can comprise those nucleotides of SEQ ID NO: 1 or SEQID NO: 4 which encode a UCH-1, UCH-2, UBA, or UIM domain of human 23479 or 48120. The nucleotide sequence determined from the cloning of the 23479 or 48120 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 23479 or 48120 family members, or fragments thereof, as well as 23479 or 48120 homologues, or fragments thereof, from other species.

[0187] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 20 amino acids in length. Preferably, fragments are at least 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[0188] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 23479 or 48120 nucleic acid fragment can include a sequence corresponding to a UCH-1 domain and a UCH-2 domain. A 48120 nucleic acid fragment can further include a sequence corresponding to a UBA domain and a UIM domain.

[0189] 23479 or 48120 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.

[0190] In one embodiment, the probe or primer is attached to a solid support, e.g., a solid support described herein.

[0191] One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g., the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 2 or SEQ ID NO: 5. The reverse primer can anneal to the ultimate codon, e.g., the codon immediately before the stop codon, e.g., the codon encoding amino acid residue 934 of SEQ ID NO: 2 or amino acid residue 1139 of SEQ ID NO: 5. In a preferred embodiment, the annealing temperatures of the forward and reverse primers differ by no more than 5, 4, 3, or 2° C.

[0192] In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0193] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a UCH-1 domain from about amino acids about amino acid 296-327 of SEQ ID NO: 2 or amino acid 162-193 of SEQ ID NO: 5; a UCH-2 domain from about amino acid 546-640 of SEQ ID NO: 2 or amino acid 580-649 of SEQ ID NO: 5; a UBA domain from about amino acid 20-61 of SEQ ID NO: 5; or a UIM domain from about amino acid 96-113 of SEQ ID NO: 5.

[0194] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 23479 or 48120 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: a UCH-1 domain from about amino acids about amino acid 296-327 of SEQ ID NO: 2 or amino acid 162-193 of SEQ ID NO: 5; a UCH-2 domain from about amino acid 546-640 of SEQ ID NO: 2 or amino acid 580-649 of SEQ ID NO: 5; a UBA domain from about amino acid 20-61 of SEQ ID NO: 5; and a UIM domain from about amino acid 96-113 of SEQ ID NO: 5.

[0195] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[0196] A nucleic acid fragment encoding a “biologically active portion of a 23479 or 48120 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6, which encodes a polypeptide having a 23479 or 48120 biological activity (e.g., the biological activities of the 23479 or 48120 proteins are described herein), expressing the encoded portion of the 23479 or 48120 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 23479 or 48120 protein. For example, a nucleic acid fragment encoding a biologically active portion of 23479 or 48120 includes a UCH- 1 domain from about amino acids about amino acid 296-327 of SEQ ID NO: 2 or amino acid 162-193 of SEQ ID NO: 5, a UCH-2 domain from about amino acid 546-640 of SEQ ID NO: 2 or amino acid 580-649 of SEQ ID NO: 5, a UTBA domain from about amino acid 20-61 of SEQ ID NO: 5, or a UIM domain from about amino acid 96-113 of SEQ ID NO: 5. A nucleic acid fragment encoding a biologically active portion of a 23479 or 48120 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.

[0197] In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6.

[0198] In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from a sequence described in WO01/55301, WO 01/57058, or WO 01/38543, or Genbank™ accession numbers AB018272 or AK001193. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 1 or SEQ ID NO: 3 located outside the region of nucleotides 2314-3363, 1788-2315, 1865-2831, 2660-3206, 1723-2649, 441-1046, 3012-3206, 2493-2726, or 1723-2372 of SEQ ID NO: 1; include one or more nucleotides from SEQ ID NO: 4 or SEQ ID NO: 6 located outside the region of nucleotides 2722-4329, 2818-3713, 1366-2233, 2923-3535, 1366-1829, 2766-3169, 2766-3962, or 3958-4653 of SEQ ID NO: 4; not include all of the nucleotides of a sequence of WO01/55301 or WO 01/57058, or Genbank™ accession numbers AB018272 or AK00193, e.g., can be one or more nucleotides shorter (at one or both ends) than a sequence of WO01/55301 or WO 01/57058, or Genbank™ accession numbers AB018272 or AK001193; or can differ by one or more nucleotides in the region of overlap.

[0199] 46689 Nucleic Acid Fragments

[0200] A nucleic acid molecule of the invention can include only a portion of the nucleic acid sequence of SEQ ID NO: 7 or 3. For example, such a nucleic acid molecule can include a fragment that can be used as a probe or primer or a fragment encoding a portion of a 46689 protein, e.g., an immunogenic or biologically active portion of a 46689 protein. A fragment can comprise those nucleotides of SEQ ID NO: 7 which encode an α/β hydrolase domain of human 46689. The nucleotide sequence determined from the cloning of the 46689 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 46689 family members, or fragments thereof, as well as 46689 homologues, or fragments thereof, from other species.

[0201] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5′ or 3′ noncoding region. Other embodiments include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50, 100, 124, 136, 150, 185, 190, 200, 238, or more amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.

[0202] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 46689 nucleic acid fragment can include a sequence corresponding to an α/β hydrolase domain, e.g., about nucleotides 670 to 1371 of SEQ ID NO: 7, a region that includes a transmembrane domain, e.g., about nucleotides 115 to 669 of SEQ ID NO: 7, or a region that includes both an α/β hydrolase domain and a transmembrane domain, e.g., about nucleotides 562 to 1371 of SEQ ID NO: 7.

[0203] 46689 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 7 or SEQ ID NO: 9. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.

[0204] In one embodiment, the probe or primer is attached to a solid support, e.g., a solid support described herein.

[0205] One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g., the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 8. The reverse primer can anneal to the ultimate codon, e.g., the codon immediately before the stop codon, e.g., the codon encoding amino acid residue 468 of SEQ ID NO: 8. In a preferred embodiment, the annealing temperatures of the forward and reverse primers differ by no more than 5, 4, 3, or 2° C.

[0206] In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0207] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: an α/β hydrolase domain, e.g., about amino acid residues 186 to 419 of SEQ ID NO: 8; a region that includes a transmembrane domain, e.g., about amino acid residues 1 to 185 or 27 to 185 of SEQ ID NO: 8; or a region that includes both an α/β hydrolase domain and a transmembrane domain, e.g., about amino acid residues 150 to 419 of SEQ ID NO: 8.

[0208] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 46689 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any of the following regions are provided: an α/β hydrolase domain, e.g., about amino acid residues 186 to 419 of SEQ ID NO: 8; a region that includes a transmembrane domain, e.g., about amino acid residues 1 to 185 or 27 to 185 of SEQ ID NO: 8; or a region that includes both an α/β hydrolase domain and a transmembrane domain, e.g., about amino acid residues 150 to 419 of SEQ ID NO: 8.

[0209] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[0210] A nucleic acid fragment encoding a “biologically active portion of a 46689 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 7 or 3, which encodes a polypeptide having a 46689 biological activity (e.g., the biological activities of the 46689 proteins are described herein), expressing the encoded portion of the 46689 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the 46689 protein. For example, a nucleic acid fragment encoding a biologically active portion of 46689 includes an α/β hydrolase domain, e.g., amino acid residues about 186 to 419 of SEQ ID NO: 8. A nucleic acid fragment encoding a biologically active portion of a 46689 polypeptide, may comprise a nucleotide sequence which is greater than 712 or more nucleotides in length.

[0211] In preferred embodiments, a nucleic acid includes a nucleotide sequence that is about 300, 372,408,500,555,561, 600,700,712,800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2050, or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 7, or SEQ ID NO: 9.

[0212] In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of SEQ ID NO: 7258 of WO 01/57188 or SEQ ID NO: 790 or WO 00/52165. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 1 or SEQ ID NO: 9 located outside the region of nucleotides 246 to 799, 808 to 1519, 743 to 1113, 1115 to 1521, or 1523 to 2082; not include all of the nucleotides of SEQ ID NO: 7258 of WO 01/57188 or SEQ ID NO: 790 or WO 00/52165, e.g., can be one or more nucleotides shorter (at one or both ends) than the sequence of SEQ ID NO: 7258 of WO 01/57188 or SEQ ID NO: 790 or WO 00/52165; or can differ by one or more nucleotides in the region of overlap.

[0213] 23479, 48120, or 46689 Nucleic Acid Variants

[0214] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9. Such differences can be due to degeneracy of the genetic code (and result in a nucleic acid which encodes the same 23479, 48120, or 46689 proteins as those encoded by the nucleotide sequence disclosed herein). In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. If alignment is needed for this comparison the sequences should be aligned for maximum homology. The encoded protein can differ by no more than 5, 4, 3, 2, or 1 amino acid. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0215] Nucleic acids of the inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% of the codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[0216] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[0217] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of the nucleotides in the subject nucleic acid. The nucleic acid can differ by no more than 5, 4, 3, 2, or 1 nucleotide. If necessary for this analysis the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.

[0218] Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO 2 or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of the 23479, 48120, or 46689 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 23479, 48120, or 46689 gene.

[0219] Preferred variants include those that are correlated with hydrolase activity, e.g., the hydrolysis of a substrate molecule, e.g., a protein (e.g., a ubiquitinated protein or poly-ubiquitin), lipid, or small molecule (e.g., metabolite, signaling molecule, toxin, or carcinogen) substrate.

[0220] Allelic variants of 23479, 48120, or 46689, e.g., human 23479, 48120, or 46689, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the 23479, 48120, or 46689 protein within a population that maintain the ability to bind and hydrolyze substrate molecules, e.g., protein (e.g., a ubiquitinated protein or poly-ubiquitin), lipid, or small molecule (e.g., metabolite, signaling molecule, toxin, or carcinogen) substrates. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants of the 23479, 48120, or 46689, e.g., human 23479, 48120, or 46689, protein within a population that do not have the ability to bind and hydrolyze substrate molecules, e.g., protein (e.g., a ubiquitinated protein or poly-ubiquitin), lipid, or small molecule (e.g., metabolite, signaling molecule, toxin, or carcinogen) substrates. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, or a substitution, insertion, or deletion in critical residues or critical regions of the protein.

[0221] Moreover, nucleic acid molecules encoding other 23479, 48120, or 46689 family members and, thus, which have a nucleotide sequence which differs from the 23479, 48120, or 46689 sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9 are intended to be within the scope of the invention.

[0222] Antisense Nucleic Acid Molecules, Ribozymes and Modified 23479, 48120, or 46689 Nucleic Acid Molecules

[0223] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 23479, 48120, or 46689. An “antisense” nucleic acid can include a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 23479, 48120, or 46689 coding strand, or to only a portion thereof (e.g., the coding region of human 23479, 48120, or 46689 corresponding to SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 9, respectively). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding 23479, 48120, or 46689 (e.g., the 5′ and 3′ untranslated regions).

[0224] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 23479, 48120, or 46689 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of 23479, 48120, or 46689 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 23479, 48120, or 46689 mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[0225] An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0226] The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 23479, 48120, or 46689 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0227] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0228] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a 23479, 48120, or 46689-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 23479, 48120, or 46689 cDNA disclosed herein (i.e., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a 23479, 48120, or 46689-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, 23479, 48120, or 46689 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0229]23479, 48120, or 46689 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the 23479, 48120, or 46689 (e.g., the 23479, 48120, or 46689 promoter and/or enhancers) to form triple helical structures that prevent transcription of the 23479, 48120, or 46689 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[0230] The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or calorimetric.

[0231] A 23479, 48120, or 46689 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulmé (2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense agents.

[0232] For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675. PNAs of 23479, 48120, or 46689 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 23479, 48120, or 46689 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0233] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0234] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 23479, 48120, or 46689 nucleic acid of the invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence of the 23479, 48120, or 46689 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

[0235] Isolated 23479 or 48120 Polypeptides

[0236] In another aspect, the invention features an isolated 23479 or 48120 protein, or fragment, e.g., a biologically active portion, for use as immunogens Ur antigens to raise or test (or more generally to bind) anti-23479 or 48120 antibodies. 23479 or 48120 protein can be isolated from cells or tissue sources using standard protein purification techniques. 23479 or 48120 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[0237] Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[0238] In a preferred embodiment, a 23479 or 48120 polypeptide has one or more of the following characteristics:

[0239] (i) it has the ability to cleave a bond between ubiquitin and a substrate, e.g., a protein targeted for degradation by ubiquitination;

[0240] (ii) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of a 23479 or 48120 polypeptide, e.g., a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 5;

[0241] (iii) it has an overall sequence similarity of at least 60%, more preferably at least 70%, 80%, 90%, 95%, 98%, 99%, or more with a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 5;

[0242] (iv) it has a UCH- 1 domain which preferably has an overall sequence similarity of at least about 70%, 80%, 90% or 95% with amino acid residues about 296-327 of SEQ ID NO: 2 or amino acid 162-193 of SEQ ID NO: 5;

[0243] (v) it has a UCH-2 domain, or region which has an overall sequence similarity of at least about 70%, 80%, 90% or 95% with amino acid residues about 546-640 of SEQ ID NO: 2 or amino acid 580-649 of SEQ ID NO: 5;

[0244] (vi) it has a UBA domain, or region which has an overall sequence similarity of at least about 70%, 80%, 90% or 95% with amino acid residues about 20-61 of SEQ ID NO: 5;

[0245] (vii) it has a UIM domain, or region which has an overall sequence similarity of at least about 70%, 80%, 90% or 95% with amino acid residues about 20-61 of SEQ ID NO: 5; and

[0246] (viii) it has at least one, two, three, four, five, or six predicted N-glycosylation sites (PS00001);

[0247] (ix) it has at least one predicted cAMP and cGMP-dependent protein kinase phosphorylation site (PS00004);

[0248] (x) it has at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 predicted protein kinase C phosphorylation sites (PS00005);

[0249] (xi) it has at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or even 33 predicted casein kinase II phosphorylation sites (PS00006);

[0250] (xii) it has at least one, two, or three predicted tyrosine kinase phosphorylation sites (PS00007);

[0251] (xiii) it has at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, or even 13 predicted N-myristoylation sites (PS00008);

[0252] (xiv) it has at least one amidation site (PS00009);

[0253] (xv) it has at least one carbamoyl-phosphate synthase subdomain signature 2 (PS00867);

[0254] (xvi) at least one ubiquitin carboxyl-terminal hydrolase family 2 signature 2 (PS00973);

[0255] (xvii) it has at least one predicted peroxisomal targeting signal; and

[0256] (xviii) it has at least one coiled coil domain.

[0257] In a preferred embodiment the 23479 or 48120 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO: 2 or SEQ ID NO: 5. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 2 or SEQ ID NO: 5 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 2 or SEQ ID NO: 5. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the UCH-1, UCH-2, UBA, or UIM domains. In another preferred embodiment one or more differences are in the UCH- 1, UCH-2, UBA, or UIM domains.

[0258] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 23479 or 48120 proteins differ in amino acid sequence from SEQ ID NO: 2 or SEQ ID NO: 5, yet retain biological activity.

[0259] In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 2 or SEQ ID NO: 5.

[0260] A 23479 protein or fragment is provided which varies from the sequence of SEQ ID NO: 2 in regions defined by amino acids about 1-295, 328-545, or 641-934 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 2 in regions defined by amino acids about 296-327 or 546-640. A 48120 protein or fragment is provided which varies from the sequence of SEQ ID NO: 5 in regions defined by amino acids about 1-19, 62-95, 114-161, 194-579, or 650-1139 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 5 in regions defined by amino acids about 20-61, 96-113, 162-193, or 580-649. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[0261] In one embodiment, a biologically active portion of a 23479 or 48120 protein includes a UCH-1, UCH-2, UBA, or UIM domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 23479 or 48120 protein.

[0262] In a preferred embodiment, the 23479 or 48120 protein has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5. In other embodiments, the 23479 or 48120 protein is substantially identical to SEQ ID NO: 2 or SEQ ID NO: 5. In yet another embodiment, the 23479 or 48120 protein is substantially identical to SEQ ID NO: 2 or SEQ ID NO: 5 and retains the functional activity of the protein of SEQ ID NO: 2 or SEQ ID NO: 5, as described in detail in the subsections above.

[0263] In a preferred embodiment, a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in WO01/55301, WO 01/57058, WO 01/57272, or WO 01/38543, or Genbank™ accession number BAA34449. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO: 2 or SEQ ID NO: 5 outside the region of amino acid residues 488-843, 638-934, 753-934, or 440-510 of SEQ ID NO: 2 or 428-716, 880-1139, 912-1139, 428-582, 1017-1081 of SEQ ID NO: 5; not include all of the amino acid residues of a sequence present in WO01/55301, WO 01/57058, WO 01/57272, or WO 01/38543, or Genbank™ accession number BAA344449, e.g., can be one or more amino acid residues shorter (at one or both ends) than a sequence present in WO01/55301, WO 01/57058, WO 01/57272, or WO 01/38543, or Genbank™ accession number BAA344449; or can differ by one or more amino acid residues in the region of overlap.

[0264] Isolated 46689 Polypeptides

[0265] In another aspect, the invention features, an isolated 46689 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-46689 antibodies. 46689 protein can be isolated from cells or tissue sources using standard protein purification techniques. 46689 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.

[0266] Polypeptides of the invention include those that arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell.

[0267] In a preferred embodiment, a 46689 polypeptide has one or more of the following characteristics:

[0268] (i) it has the ability to bind to and hydrolyze substrate molecules, e.g., protein, lipid, or small molecule (e.g., metabolite, signaling molecule, toxin, or carcinogen) substrates;

[0269] (ii) it has a molecular weight, e.g., a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of a 46689 polypeptide, e.g., a polypeptide of SEQ ID NO: 8;

[0270] (iii) it has an overall sequence similarity of at least 60%, more preferably at least 70%, 80%, 90%, 95%, 98%, 99%, or more with a polypeptide a of SEQ ID NO: 8;

[0271] (iv) it can be found in tumors;

[0272] (v) it has an alp hydrolase domain which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more identical with amino acid residues about 186 to 419 of SEQ ID NO: 8;

[0273] (vi) it has a catalytic acid residue;

[0274] (vii) it has a catalytic histidine residue;

[0275] (viii) it has a transmembrane domain, or a region which is about 70%, 80%, 90%, 95%, 98%, 99%, or more identical with amino acid residues about 150 to 167 or SEQ ID NO: 8;

[0276] (ix) it has a signal peptide, or a region which is about 70%, 80%, 90%, 95%, 98%, 99%, or more identical with amino acid residues about 150 to 167 or SEQ ID NO: 8;

[0277] (x) it has at least one, two, three, preferably four predicted protein kinase C phosphorylation sites (PS00005);

[0278] (xi) it has at least one, two, three, preferably four predicted casein kinase II phosphorylation sites (PS00006);

[0279] (xii) it has at least one predicted cAMP- and cGMP-dependent protein kinase phosphorylation site (PS00004);

[0280] (xiii) it has at least one, preferably two predicted amidation sites (PS00009); and

[0281] (xiv) it has at least one, two, three, four, preferably five predicted N-myristylation sites (PS00008).

[0282] In a preferred embodiment the 46689 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID: 2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 8 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO: 8. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In a preferred embodiment the differences are not in the α/β hydrolase domain, e.g., about amino acid residues 186 to 419 of SEQ ID NO: 8. or the transmembrane domain, e.g., about amino acid residues 150 to 167 of SEQ ID NO: 8. In another preferred embodiment one or more differences are in the α/β hydrolase domain, e.g., about amino acid residues 186 to 419 of SEQ ID NO: 8. or the transmembrane domain, e.g., about amino acid residues 150 to 167 of SEQ ID NO: 8

[0283] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 46689 proteins differ in amino acid sequence from SEQ ID NO: 8, yet retain biological activity.

[0284] In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 8.

[0285] A 46689 protein or fragment is provided which varies from the sequence of SEQ ID NO: 8 in regions defined by amino acid residues about 27 to 149, 168 to 185, 242 to 350, and 400 to 468 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 8 in regions defined by amino acids about 150 to 167, 186 to 241, and 351 to 399. (If this comparison requires alignment the sequences should be aligned for maximum homology. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.

[0286] In one embodiment, a biologically active portion of a 46689 protein includes an α/β hydrolase domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native 46689 protein.

[0287] In a preferred embodiment, the 46689 protein has an amino acid sequence shown in SEQ ID NO: 8. In other embodiments, the 46689 protein is substantially identical to SEQ ID NO: 8. In yet another embodiment, the 46689 protein is substantially identical to SEQ ID NO: 8 and retains the functional activity of the protein of SEQ ID NO: 8, as described in detail in the subsections above.

[0288] In a preferred embodiment, a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in SEQ ID NO: 7258 of WO 01/57188 or SEQ ID NO: 790 or WO 00/52165. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO: 2 outside the region encoded by nucleotides 246 to 799, 808 to 1519, 743 to 1113, 1115 to 1521, or 1523 to 2082 of SEQ ID NO: 7; not include all of the amino acid residues encoded by a nucleotide sequence in SEQ ID NO: 7258 of WO 01/57188 or SEQ ID NO: 790 or WO 00/52165, e.g., can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by a nucleotide sequence in SEQ ID NO: 7258 of WO 01/57188 or SEQ ID NO: 790 or WO 00/52165; or can differ by one or more amino acid residues in the region of overlap.

[0289] 23479, 48120, or 46689 Chimeric or Fusion Proteins

[0290] In another aspect, the invention provides 23479, 48120, or 46689 chimeric or fusion proteins. As used herein, a 23479, 48120, or 46689 “chimeric protein” or “fusion protein” includes a 23479, 48120, or 46689 polypeptide linked to a non-23479, 48120, or 46689 polypeptide. A “non-23479, 48120, or 46689 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 23479, 48120, or 46689 protein, e.g., a protein which is different from the 23479, 48120, or 46689 protein and which is derived from the same or a different organism. The 23479, 48120, or 46689 polypeptide of the fusion protein can correspond to all or a portion e.g., a fragment described herein of a 23479, 48120, or 46689 amino acid sequence. In a preferred embodiment, a 23479, 48120, or 46689 fusion protein includes at least one (or two) biologically active portion of a 23479, 48120, or 46689 protein. The non-23479, 48120, or 46689 polypeptide can be fused to the N-terminus or C-terminus of the 23479, 48120, or 46689 polypeptide.

[0291] The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-23479, 48120, or 46689 fusion protein in which the 23479, 48120, or 46689 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant 23479, 48120, or 46689. Alternatively, the fusion protein can be a 23479, 48120, or 46689 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 23479, 48120, or 46689 can be increased through use of a heterologous signal sequence.

[0292] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albunin.

[0293] The 23479, 48120, or 46689 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 23479, 48120, or 46689 fusion proteins can be used to affect the bioavailability of a 23479, 48120, or 46689 substrate. 23479, 48120, or 46689 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 23479, 48120, or 46689 protein; (ii) mis-regulation of the 23479, 48120, or 46689 gene; and (iii) aberrant post-translational modification of a 23479, 48120, or 46689 protein.

[0294] Moreover, the 23479, 48120, or 46689-fusion proteins of the invention can be used as immunogens to produce anti-23479, 48120, or 46689 antibodies in a subject, to purify 23479, 48120, or 46689 ligands and in screening assays to identify molecules which inhibit the interaction of 23479, 48120, or 46689 with a 23479, 48120, or 46689 substrate.

[0295] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 23479, 48120, or 46689-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 23479, 48120, or 46689 protein.

[0296] Variants of 23479, 48120, or 46689 Proteins

[0297] In another aspect, the invention also features a variant of a 23479, 48120, or 46689 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants of the 23479, 48120, or 46689 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 23479, 48120, or 46689 protein. An agonist of the 23479, 48120, or 46689 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a 23479, 48120, or 46689 protein. An antagonist of a 23479, 48120, or 46689 protein can inhibit one or more of the activities of the naturally occurring form of the 23479, 48120, or 46689 protein by, for example, competitively modulating a 23479, 48120, or 46689-mediated activity of a 23479, 48120, or 46689 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the 23479, 48120, or 46689 protein.

[0298] Variants of a 23479, 48120, or 46689 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 23479, 48120, or 46689 protein for agonist or antagonist activity.

[0299] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a 23479, 48120, or 46689 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 23479, 48120, or 46689 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[0300] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of 23479, 48120, or 46689 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 23479, 48120, or 46689 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[0301] Cell based assays can be exploited to analyze a variegated 23479, 48120, or 46689 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cell line, which ordinarily responds to 23479, 48120, or 46689 in a substrate-dependent manner. The transfected cells are then contacted with 23479, 48120, or 46689 and the effect of the expression of the mutant on signaling by the 23479, 48120, or 46689 substrate can be detected, e.g., by measuring changes in cellular proliferation. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 23479, 48120, or 46689 substrate, and the individual clones further characterized.

[0302] In another aspect, the invention features a method of making a 23479, 48120, or 46689 polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 23479, 48120, or 46689 polypeptide, e.g., a naturally occurring 23479, 48120, or 46689 polypeptide. The method includes: altering the sequence of a 23479, 48120, or 46689 polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[0303] In another aspect, the invention features a method of making a fragment or analog of a 23479, 48120, or 46689 polypeptide a biological activity of a naturally occurring 23479, 48120, or 46689 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 23479, 48120, or 46689 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

[0304] Anti-23479, 48120, or 46689 Antibodies

[0305] In another aspect, the invention provides an anti-23479, 48120, or 46689 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0306] The anti-23479, 48120, or 46689 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0307] As used herein, the term “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[0308] The term “antigen-binding fragment” of an antibody (or simply “antibody portion,” or “fragment”), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 23479, 48120, or 46689 polypeptide or fragment thereof. Examples of antigen-binding fragments of the anti-23479, 48120, or 46689 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0309] The anti-23479, 48120, or 46689 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[0310] Phage display and combinatorial methods for generating anti-23479, 48120, or 46689 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

[0311] In one embodiment, the anti-23479, 48120, or 46689 antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art.

[0312] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

[0313] An anti-23479, 48120, or 46689 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

[0314] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0315] A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding of the humanized antibody to a 23479, 48120, or 46689 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

[0316] As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

[0317] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and and 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 23479, 48120, or 46689 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[0318] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

[0319] Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues of the humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[0320] In preferred embodiments an antibody can be made by immunizing with purified 23479, 48120, or 46689 antigen, or a fragment thereof, e.g., a fragment described herein, membrane associated antigen, tissue, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g., cytosol or membrane fractions.

[0321] A full-length 23479, 48120, or 46689 protein or, antigenic peptide fragment of 23479, 48120, or 46689 can be used as an immunogen or can be used to identify anti-23479, 48120, or 46689 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. The antigenic peptide of 23479, 48120, or 46689 should include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 and encompasses an epitope of 23479, 48120, or 46689. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0322] Fragments of 23479 or 48120 can be used, e.g., to characterize the specificity of an antibody or to make immunogens. For example, fragments of 23479 or 48120 which include residues about 275 to 290, 530 to 550, or 640 to 650 of SEQ ID NO: 2 or residues about 120 to 155, 680 to 700, or 770 to 800 of SEQ ID NO: 5 can be used to make antibodies against hydrophilic regions of the 23479 or 48120 protein. Similarly, fragments of 23479 or 48120 which include residues about 100 to 110, 295 to 310, or 920 to 930 of SEQ ID NO: 2 or residues about 1040 to 1055 of SEQ ID NO: 5 can be used to make an antibody against a hydrophobic region of the 23479 or 48120 protein; a fragment of 23479 or 48120 which includes residues about 296-327 of SEQ ID NO: 2 or 162-193 of SEQ ID NO: 5 can be used to make an antibody against the UCH-1 region of the 23479 or 48120 protein; a fragment of 23479 or 48120 which includes residues about 546-640 of SEQ ID NO: 2 or 580-649 of SEQ ID NO: 5 can be used to make an antibody against the UCH-2 region of the 23479 or 48120 protein; a fragment of 48120 which includes residues about 20-61 of SEQ ID NO: 5 can be used to make an antibody against the UBA region of the 48120 protein; and a fragment of 48120 which includes residues about 96-113 of SEQ ID NO: 5 can be used to make an antibody against the UIM region of the 48120 protein.

[0323] Similarly, fragments of 46689 can be used, e.g., to characterize the specificity of an antibody or to make immunogens. For example, fragments of 46689 which include residues about 34 to 51, about 333 to 347, or about 438 to 449 of SEQ ID NO: 8 can be used to make antibodies against hydrophilic regions of the 46689 protein. Similarly, fragments of 46689 which include residues about 1 to 23, about 133 to 145, or about 150 to 168 of SEQ ID NO: 8 can be used to make an antibody against a hydrophobic region of the 46689 protein; fragments of 46689 which include residues about 27 to 149 or about 168 to 468 of SEQ ID NO: 8 can be used to make an antibody against an non-transmembrane region of the 46689 protein; or fragments of 46689 which include residues about 186 to 241 or about 350 to 400 of SEQ ID NO: 8 can be used to make an antibody against the α/β hydrolase domain of the 46689 protein.

[0324] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[0325] Antibodies that bind only native 23479, 48120, or 46689 protein, only denatured or otherwise non-native 23479, 48120, or 46689 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies that bind to native, but not denatured 23479, 48120, or 46689 protein.

[0326] Preferred epitopes encompassed by the antigenic peptide are regions of 23479, 48120, or 46689 are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human 23479, 48120, or 46689 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the 23479, 48120, or 46689 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[0327] In a preferred embodiment the antibody can bind to the extracellular portion of the 46689 protein, e.g., it can bind to a whole cell which expresses the 46689 protein. In another embodiment, the antibody binds an intracellular portion of the 46689 protein.

[0328] In preferred embodiments, antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g., tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g., cytosol or membrane fractions.

[0329] The anti-23479, 48120, or 46689 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target 23479, 48120, or 46689 protein.

[0330] In a preferred embodiment the antibody has effector function and/or can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement.

[0331] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[0332] In a preferred embodiment, an anti-23479 or 48120 antibody alters (e.g., increases or decreases) the de-ubiquitination activity of a 23479 or 48120 polypeptide. For example, the antibody can bind at or in proximity to the active site, e.g., to an epitope that includes a residue located from about 550-567 of SEQ ID NO: 2 or 584-601 of SEQ ID NO: 5.

[0333] In another preferred embodiment, an anti-46689 antibody alters (e.g., increases or decreases) the hydrolase activity of a 46689 polypeptide. For example, the antibody can bind at or in proximity to the active site, e.g., to an epitope that includes a residue located from about 186 to 241 or 350 to 400 of SEQ ID NO: 8.

[0334] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[0335] An anti-23479, 48120, or 46689 antibody (e.g., monoclonal antibody) can be used to isolate 23479, 48120, or 46689 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-23479, 48120, or 46689 antibody can be used to detect 23479, 48120, or 46689 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein. Anti-23479, 48120, or 46689 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0336] The invention also includes a nucleic acid which encodes an anti-23479, 48120, or 46689 antibody, e.g., an anti-23479, 48120, or 46689 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells.

[0337] The invention also includes cell lines, e.g., hybridomas, which make an anti-23479, 48120, or 46689 antibody, e.g., an antibody described herein, and method of using said cells to make a 23479, 48120, or 46689 antibody.

[0338] Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells

[0339] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.

[0340] A vector can include a 23479, 48120, or 46689 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 23479, 48120, or 46689 proteins, mutant forms of 23479, 48120, or 46689 proteins, fusion proteins, and the like).

[0341] The recombinant expression vectors of the invention can be designed for expression of 23479, 48120, or 46689 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0342] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Purified fusion proteins can be used in 23479, 48120, or 46689 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 23479, 48120, or 46689 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector of the present invention can be used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six weeks).

[0343] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0344] The 23479, 48120, or 46689 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian cells.

[0345] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.

[0346] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).

[0347] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0348] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[0349] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 23479, 48120, or 46689 nucleic acid molecule within a recombinant expression vector or a 23479, 48120, or 46689 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0350] A host cell can be any prokaryotic or eukaryotic cell. For example, a 23479, 48120, or 46689 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) CellI23:175-182)). Other suitable host cells are known to those skilled in the art.

[0351] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[0352] A host cell of the invention can be used to produce (i.e., express) a 23479, 48120, or 46689 protein. Accordingly, the invention further provides methods for producing a 23479, 48120, or 46689 protein using the host cells of the invention. In one embodiment, the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a 23479, 48120, or 46689 protein has been introduced) in a suitable medium such that a 23479, 48120, or 46689 protein is produced. In another embodiment, the method further includes isolating a 23479, 48120, or 46689 protein from the mediumn or the host cell.

[0353] In another aspect, the invention features, a cell or purified preparation of cells which include a 23479, 48120, or 46689 transgene, or which otherwise misexpress 23479, 48120, or 46689. The cell preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a 23479, 48120, or 46689 transgene, e.g., a heterologous form of a 23479, 48120, or 46689, e.g., a gene derived from humans (in the case of a non-human cell). The 23479, 48120, or 46689 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 23479, 48120, or 46689, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 23479, 48120, or 46689 alleles or for use in drug screening.

[0354] In another aspect, the invention features, a human cell, e.g., a hematopoietic or hepatic stem cell, transformed with nucleic acid which encodes a subject 23479, 48120, or 46689 polypeptide.

[0355] Also provided are cells, preferably human cells, e.g., human hematopoietic, hepatic, neural, lung, ovary, breast or fibroblast cells, in which an endogenous 23479, 48120, or 46689 is under the control of a regulatory sequence that does not normally control the expression of the endogenous 23479, 48120, or 46689 gene. The expression characteristics of an endogenous gene within a cell, e.g., a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous 23479, 48120, or 46689 gene. For example, an endogenous 23479, 48120, or 46689 gene which is “transcriptionally silent,” e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

[0356] In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 23479, 48120, or 46689 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki etal. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No. 5,876,742. Production of 23479, 48120, or 46689 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 23479, 48120, or 46689 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

[0357] Transgenic Animals

[0358] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 23479, 48120, or 46689 protein and for identifying and/or evaluating modulators of 23479, 48120, or 46689 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 23479, 48120, or 46689 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0359] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of a 23479, 48120, or 46689 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 23479, 48120, or 46689 transgene in its genome and/or expression of 23479, 48120, or 46689 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 23479, 48120, or 46689 protein can further be bred to other transgenic animals carrying other transgenes. 23479, 48120, or 46689 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[0360] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

[0361] Uses

[0362] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).

[0363] The isolated nucleic acid molecules of the invention can be used, for example, to express a 23479, 48120, or 46689 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect a 23479, 48120, or 46689 mRNA (e.g., in a biological sample) or a genetic alteration in a 23479, 48120, or 46689 gene, and to modulate 23479, 48120, or 46689 activity, as described further below. The 23479, 48120, or 46689 proteins can be used to treat disorders characterized by insufficient or excessive production of a 23479, 48120, or 46689 substrate or production of 23479, 48120, or 46689 inhibitors. In addition, the 23479, 48120, or 46689 proteins can be used to screen for naturally occurring 23479, 48120, or 46689 substrates, to screen for drugs or compounds which modulate 23479, 48120, or 46689 activity, as well as to treat disorders characterized by insufficient or excessive production of 23479, 48120, or 46689 protein or production of 23479, 48120, or 46689 protein forms which have decreased, aberrant or unwanted activity compared to 23479, 48120, or 46689 wild type protein (e.g., a cellular proliferative or differentiative disorder, e.g., in the lung, brain, ovary, or breast; a neural disorder; a hematopoietic disorder; a cardiovascular disorder; or a liver disorder). Moreover, the anti-23479, 48120, or 46689 antibodies of the invention can be used to detect and isolate 23479, 48120, or 46689 proteins, regulate the bioavailability of 23479, 48120, or 46689 proteins, and modulate 23479, 48120, or 46689 activity.

[0364] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 23479, 48120, or 46689 polypeptide is provided. The method includes: contacting the compound with the subject 23479, 48120, or 46689 polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject 23479, 48120, or 46689 polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 23479, 48120, or 46689 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 23479, 48120, or 46689 polypeptide. Screening methods are discussed in more detail below.

[0365] Screening Assays

[0366] The invention provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 23479, 48120, or 46689 proteins, have a stimulatory or inhibitory effect on, for example, 23479, 48120, or 46689 expression or 23479, 48120, or 46689 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 23479, 48120, or 46689 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 23479, 48120, or 46689 genes) in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt normal target gene interactions.

[0367] In one embodiment, the invention provides assays for screening candidate or test compounds that are substrates of a 23479, 48120, or 46689 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 23479, 48120, or 46689 protein or polypeptide or a biologically active portion thereof.

[0368] In one embodiment, an activity of a 23479 or 48120 protein can be assayed by measuring or detecting 23479- or 48120-mediated de-ubiquitination. De-ubiquitination assays useful for detecting a ubiquitin carboxyl-terminal hydrolase activity are well known in the art and can be found, for example, in Zhu et al. (1997) Journal of Biological Chemistry 272:51-57, Mitch et al. (1999) American Journal of Physiology 276:Cl 132-C1 138, Liu et al. (1999) Molecular and Cell Biology 19:3029-3038, and such as those cited in various reviews, for example, Ciechanover et al. (1994) The FASEB Journal 8:182-192, Chiechanover (1994) Biol. Chem. Hoppe-Seyler 375:565-581, Hershko et al. (1998) Annual Review of Biochemistry 67:425-479, Swartz (1999) Annual Review of Medicine 50:57-74, Ciechanover (1998) EMBO Journal 17:7151-7160, and D'Andrea et al. (1998) Critical Reviews in Biochemistry and Molecular Biology 33:337-352. These assays include, but are not limited to, the disappearance of substrate, including a decrease in the amount of polyubiquitin or ubiquitinated substrate protein or protein remnant, appearance of intermediate and end products, such as appearance of free ubiquitin monomers, general protein turnover, specific protein turnover, ubiquitin binding, binding to ubiquitinated substrate protein, subunit interaction, interaction with ATP, interaction with cellular components such as trans-acting regulatory factors, stabilization of specific proteins, and the like.

[0369] In one embodiment, an activity of a 46689 protein can be assayed in vitro. First, 46689 protein can be expressed in a bacterial cell and then purified, e.g., by means of an covalently attached affinity tag, e.g., a His-6 tag. Purified 46689 can then be incubated in buffer, e.g., 100 mM Tris-HCL, pH 8.5, along with a substrate molecule, e.g., a protein, lipid or small molecule, e.g., steroid, signaling molecule, toxin, or carcinogen, substrate. By measuring the optical density of the solution at various time points, the loss of substrate or increase in product can be monitored, which is a direct measure of 46689 activity. The appropriate wavelength used to measure optical density will depend upon the light absorption spectra of the substrate and product molecules. An example of such as assay, used to monitor the activity of a 2-Hydroxymuconic Semialdehyde Dehydrogenase enzyme, is provided in Inoue et al. (1995), J of Bacteriology 177(5):1196-1201, the contents of which are incorporated herein by reference.

[0370] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrugDes. 12:145).

[0371] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J Med. Chem. 37:2678; Cho et al (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

[0372] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[0373] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 23479, 48120, or 46689 protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate 23479, 48120, or 46689 activity is determined. Determining the ability of the test compound to modulate 23479, 48120, or 46689 activity can be accomplished by monitoring, for example, hydrolase activity, e.g., the hydrolysis of a substrate, e.g., a protein (e.g., a ubiquitinated protein or poly-ubiquitin), lipid, or small molecule, e.g., steroid, signaling molecule, toxin, or carcinogen, substrate. The cell, for example, can be of mammalian origin, e.g., human.

[0374] The ability of the test compound to modulate 23479, 48120, or 46689 binding to a compound, e.g., a 23479, 48120, or 46689 substrate, or to bind to 23479, 48120, or 46689 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to 23479, 48120, or 46689 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 23479, 48120, or 46689 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 23479, 48120, or 46689 binding to a 23479, 48120, or 46689 substrate in a complex. For example, compounds (e.g., 23479, 48120, or 46689 substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0375] The ability of a compound (e.g., a 23479, 48120, or 46689 substrate) to interact with 23479, 48120, or 46689 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 23479, 48120, or 46689 without the labeling of either the compound or the 23479, 48120, or 46689. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and 23479, 48120, or 46689.

[0376] In yet another embodiment, a cell-free assay is provided in which a 23479, 48120, or 46689 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the 23479, 48120, or 46689 protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the 23479, 48120, or 46689 proteins to be used in assays of the present invention include fragments which participate in interactions with non-23479, 48120, or 46689 molecules, e.g., fragments with high surface probability scores.

[0377] Soluble and/or membrane-bound forms of isolated proteins (e.g., 23479, 48120, or 46689 proteins or biologically active portions thereof) can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n), 3-[(3-cholamidopropyl) dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0378] Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[0379] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[0380] In another embodiment, determining the ability of the 23479, 48120, or 46689 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BlAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[0381] In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

[0382] It may be desirable to immobilize either 23479, 48120, or 46689, an anti-23479, 48120, or 46689 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a 23479, 48120, or 46689 protein, or interaction of a 23479, 48120, or 46689 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/23479, 48120, or 46689 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 23479, 48120, or 46689 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 23479, 48120, or 46689 binding or activity determined using standard techniques.

[0383] Other techniques for immobilizing either a 23479, 48120, or 46689 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 23479, 48120, or 46689 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

[0384] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[0385] In one embodiment, this assay is performed utilizing antibodies reactive with 23479, 48120, or 46689 protein or target molecules but which do not interfere with binding of the 23479, 48120, or 46689 protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or 23479, 48120, or 46689 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 23479, 48120, or 46689 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 23479, 48120, or 46689 protein or target molecule.

[0386] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[0387] In a preferred embodiment, the assay includes contacting the 23479, 48120, or 46689 protein or biologically active portion thereof with a known compound which binds 23479, 48120, or 46689 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 23479, 48120, or 46689 protein, wherein determining the ability of the test compound to interact with a 23479, 48120, or 46689 protein includes determining the ability of the test compound to preferentially bind to 23479, 48120, or 46689 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

[0388] The target gene products of the invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as “binding partners.” Compounds that disrupt such interactions can be useful in regulating the activity of the target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 23479, 48120, or 46689 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability of the test compound to modulate the activity of a 23479, 48120, or 46689 protein through modulation of the activity of a downstream effector of a 23479, 48120, or 46689 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

[0389] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

[0390] These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

[0391] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[0392] In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[0393] Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[0394] In an alternate embodiment of the invention, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[0395] In yet another aspect, the 23479, 48120, or 46689 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 23479, 48120, or 46689 (“23479, 48120, or 46689-binding proteins” or “23479, 48120, or 46689-bp”) and are involved in 23479, 48120, or 46689 activity. Such 23479, 48120, or 46689-bps can be activators or inhibitors of signals by the 23479, 48120, or 46689 proteins or 23479, 48120, or 46689 targets as, for example, downstream elements of a 23479, 48120, or 46689-mediated signaling pathway.

[0396] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 23479, 48120, or 46689 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: 23479, 48120, or 46689 protein can be the fused to the activator domain.) If the “bait” and the “prey” proteins are able to interact, in vivo, forming a 23479, 48120, or 46689-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional iregulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 23479, 48120, or 46689 protein.

[0397] In another embodiment, modulators of 23479, 48120, or 46689 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 23479, 48120, or 46689 mRNA or protein evaluated relative to the level of expression of 23479, 48120, or 46689 mRNA or protein in the absence of the candidate compound. When expression of 23479, 48120, or 46689 mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of 23479, 48120, or 46689 mRNA or protein expression. Alternatively, when expression of 23479, 48120, or 46689 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of 23479, 48120, or 46689 mRNA or protein expression. The level of 23479, 48120, or 46689 mRNA or protein expression can be determined by methods described herein for detecting 23479, 48120, or 46689 mRNA or protein.

[0398] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a 23479, 48120, or 46689 protein can be confirmed in vivo, e.g., in an animal such as an animal model for a cellular proliferative or differentiative disorder.

[0399] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 23479, 48120, or 46689 modulating agent, an antisense 23479, 48120, or 46689 nucleic acid molecule, a 23479, 48120, or 46689-specific antibody, or a 23479, 48120, or 46689-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

[0400] Detection Assays

[0401] Portions or fragments of the nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 23479, 48120, or 46689 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[0402] Chromosome Mapping

[0403] The 23479, 48120, or 46689 nucleotide sequences or portions thereof can be used to map the location of the 23479, 48120, or 46689 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 23479, 48120, or 46689 sequences with genes associated with disease.

[0404] Briefly, 23479, 48120, or 46689 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 23479, 48120, or 46689 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 23479, 48120, or 46689 sequences will yield an amplified fragment.

[0405] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924).

[0406] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 23479, 48120, or 46689 to a chromosomal location.

[0407] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).

[0408] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[0409] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[0410] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 23479, 48120, or 46689 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[0411] Tissue Typing

[0412] 23479, 48120, or 46689 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[0413] Furthermore, the sequences of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 23479, 48120, or 46689 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.

[0414] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 1 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0415] If a panel of reagents from 23479, 48120, or 46689 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[0416] Use of Partial 23479, 48120, or 46689 Sequences in Forensic Biology

[0417] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[0418] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO: 1 (e.g., fragments derived from the noncoding regions of SEQ ID NO: 1 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.

[0419] The 23479, 48120, or 46689 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 23479, 48120, or 46689 probes can be used to identify tissue by species and/or by organ type.

[0420] In a similar fashion, these reagents, e.g., 23479, 48120, or 46689 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[0421] Predictive Medicine

[0422] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual.

[0423] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 23479, 48120, or 46689.

[0424] Such disorders can include, e.g., a disorder associated with the misexpression of a 23479 or 48120 gene; a disorder associated with abnormal de-ubiquitination activity; and a disorder associated with abnormal protein degradation activity. Particularly preferred disorders include cellular proliferative and/or differentiative disorders, cardiovascular disorders, brain disorders. For example, preferred disorders include atherosclerosis, disorders associated with oxidative damage, cellular oxidative stress-related glucocorticoid responsiveness, and in disorders characterized by unwanted free radicals, e.g., in ischaemia reperfusion injury. Alternatively, the disorders can include, e.g., a disorder associated with the misexpression of a 46689 gene; a disorder associated with abnormal hydorlase activity, e.g., a metabolic disorder; a cellular proliferative or differentiative disorder, e.g., in the lung, brain, ovary, or breast, a neural disorder, a hematopoietic disorder, or a liver disorder.

[0425] The method includes one or more of the following:

[0426] detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the 23479, 48120, or 46689 gene, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5′ control region;

[0427] detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the 23479, 48120, or 46689 gene;

[0428] detecting, in a tissue of the subject, the misexpression of the 23479, 48120, or 46689 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

[0429] detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a 23479, 48120, or 46689 polypeptide.

[0430] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 23479, 48120, or 46689 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

[0431] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 1, or naturally occurring mutants thereof or 5′ or 3′ flanking sequences naturally associated with the 23479, 48120, or 46689 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

[0432] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the 23479, 48120, or 46689 gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of 23479, 48120, or 46689.

[0433] Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[0434] In preferred embodiments the method includes determining the structure of a 23479, 48120, or 46689 gene, an abnormal structure being indicative of risk for the disorder.

[0435] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 23479, 48120, or 46689 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.

[0436] Diagnostic and Prognostic Assays

[0437] Diagnostic and prognostic assays of the invention include method for assessing the expression level of 23479, 48120, or 46689 molecules and for identifying variations and mutations in the sequence of 23479, 48120, or 46689 molecules.

[0438] Expression Monitoring and Profiling. The presence, level, or absence of 23479, 48120, or 46689 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 23479, 48120, or 46689 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 23479, 48120, or 46689 protein such that the presence of 23479, 48120, or 46689 protein or nucleic acid is detected in the biological sample. The term “biological sample” includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression of the 23479, 48120, or 46689 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 23479, 48120, or 46689 genes; measuring the amount of protein encoded by the 23479, 48120, or 46689 genes; or measuring the activity of the protein encoded by the 23479, 48120, or 46689 genes.

[0439] The level of mRNA corresponding to the 23479, 48120, or 46689 gene in a cell can be determined both by in situ and by in vitro formats.

[0440] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 23479, 48120, or 46689 nucleic acid, such as the nucleic acid of SEQ ID NO: 1, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 23479, 48120, or 46689 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[0441] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 23479, 48120, or 46689 genes.

[0442] The level of mRNA in a sample that is encoded by one of 23479, 48120, or 46689 can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[0443] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 23479, 48120, or 46689 gene being analyzed.

[0444] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 23479, 48120, or 46689 mRNA, or genomic DNA, and comparing the presence of 23479, 48120, or 46689 mRNA or genomic DNA in the control sample with the presence of 23479, 48120, or 46689 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Pat. No. 5,695,937, is used to detect 23479, 48120, or 46689 transcript levels.

[0445] A variety of methods can be used to determine the level of protein encoded by 23479, 48120, or 46689. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[0446] The detection methods can be used to detect 23479, 48120, or 46689 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 23479, 48120, or 46689 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 23479, 48120, or 46689 protein include introducing into a subject a labeled anti-23479, 48120, or 46689 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-23479, 48120, or 46689 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[0447] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 23479, 48120, or 46689 protein, and comparing the presence of 23479, 48120, or 46689 protein in the control sample with the presence of 23479, 48120, or 46689 protein in the test sample.

[0448] The invention also includes kits for detecting the presence of 23479, 48120, or 46689 in a biological sample. For example, the kit can include a compound or agent capable of detecting 23479, 48120, or 46689 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 23479, 48120, or 46689 protein or nucleic acid.

[0449] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[0450] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[0451] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 23479, 48120, or 46689 expression or activity. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as a cellular proliferative or differentiative disorder, e.g., in the lung, brain, ovary, or breast, as well as a neural disorder, a hematopoietic disorder, a cardiovascular disorder, or a liver disorder.

[0452] In one embodiment, a disease or disorder associated with aberrant or unwanted 23479, 48120, or 46689 expression or activity is identified. A test sample is obtained from a subject and 23479, 48120, or 46689 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 23479, 48120, or 46689 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 23479, 48120, or 46689 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[0453] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 23479, 48120, or 46689 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for, e.g., a cellular proliferative or differentiative disorder.

[0454] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 23479, 48120, or 46689 in a sample, and a descriptor of the sample. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 23479, 48120, or 46689 (e.g., other genes associated with a 23479, 48120, or 46689-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).

[0455] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile of the sample, wherein the profile includes a value representing the level of 23479, 48120, or 46689 expression. The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile of the sample can be obtained by any of the methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose, e.g., a cellular proliferative or differentiative disorder, e.g., in the lung, brain, ovary, or breast, in a subject wherein an increase in 23479, 48120, or 46689 expression is an indication that the subject has or is disposed to having such a disorder. The method can be used to monitor a treatment for a disorder, e.g., a cellular proliferative or differentiative disorder, in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset of the disorder (see, e.g., Golub et al. (1999) Science 286:531).

[0456] In yet another aspect, the invention features a method of evaluating a test compound (see also, “Screening Assays”, above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 23479, 48120, or 46689 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.

[0457] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 23479, 48120, or 46689 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length of the distance vector that is the difference between the two profiles. Each of the subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile.

[0458] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison of the subject expression profile with another profile, a most similar reference profile, or a descriptor of any of the aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[0459] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 23479, 48120, or 46689 expression.

[0460] Arrays and Uses Thereof

[0461] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address of the plurality includes a capture probe that binds specifically to a 23479, 48120, or 46689 molecule (e.g., a 23479, 48120, or 46689 nucleic acid or a 23479, 48120, or 46689 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm², and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three-dimensional substrate such as a gel pad. Addresses in addition to address of the plurality can be disposed on the array.

[0462] In a preferred embodiment, at least one address of the plurality includes a nucleic acid capture probe that hybridizes specifically to a 23479, 48120, or 46689 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses of the plurality of addresses has a nucleic acid capture probe for 23479, 48120, or 46689. Each address of the subset can include a capture probe that hybridizes to a different region of a 23479, 48120, or 46689 nucleic acid. In another preferred embodiment, addresses of the subset include a capture probe for a 23479, 48120, or 46689 nucleic acid. Each address of the subset is unique, overlapping, and complementary to a different variant of 23479, 48120, or 46689 (e.g., an allelic variant, or all possible hypothetical variants). The array can be used to sequence 23479, 48120, or 46689 by hybridization (see, e.g., U.S. Pat. No. 5,695,940).

[0463] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[0464] In another preferred embodiment, at least one address of the plurality includes a polypeptide capture probe that binds specifically to a 23479, 48120, or 46689 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 23479, 48120, or 46689 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see “Anti-23479, 48120, or 46689 Antibodies,” above), such as a monoclonal antibody or a single-chain antibody.

[0465] In another aspect, the invention features a method of analyzing the expression of 23479, 48120, or 46689. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 23479, 48120, or 46689-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[0466] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 23479, 48120, or 46689. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 23479, 48120, or 46689. For example, the array can be used for the quantitation of the expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expressionper se and level of expression in that tissue.

[0467] For example, array analysis of gene expression can be used to assess the effect of cell-cell interactions on 23479, 48120, or 46689 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression.

[0468] In another embodiment, cells are contacted with a therapeutic agent. The expression profile of the cells is determined using the array, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect of the therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[0469] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 23479, 48120, or 46689-associated disease or disorder; and processes, such as a cellular transformation associated with a 23479, 48120, or 46689-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 23479, 48120, or 46689-associated disease or disorder

[0470] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including 23479, 48120, or 46689) that could serve as a molecular target for diagnosis or therapeutic intervention.

[0471] In another aspect, the invention features an array having a plurality of addresses. Each address of the plurality includes a unique polypeptide. At least one address of the plurality has disposed thereon a 23479, 48120, or 46689 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII; MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses of the plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99% identical to a 23479, 48120, or 46689 polypeptide or fragment thereof. For example, multiple variants of a 23479, 48120, or 46689 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses of the plurality. Addresses in addition to the address of the plurality can be disposed on the array.

[0472] The polypeptide array can be used to detect a 23479, 48120, or 46689 binding compound, e.g., an antibody in a sample from a subject with specificity for a 23479, 48120, or 46689 polypeptide or the presence of a 23479, 48120, or 46689-binding protein or ligand.

[0473] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 23479, 48120, or 46689 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[0474] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 23479, 48120, or 46689 or from a cell or subject in which a 23479, 48120, or 46689 mediated response has been elicited, e.g., by contact of the cell with 23479, 48120, or 46689 nucleic acid or protein, or administration to the cell or subject 23479, 48120, or 46689 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 23479, 48120, or 46689 (or does not express as highly as in the case of the 23479, 48120, or 46689 positive plurality of capture probes) or from a cell or subject which in which a 23479, 48120, or 46689 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 23479, 48120, or 46689 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody.

[0475] In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g., for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 23479, 48120, or 46689 or from a cell or subject in which a 23479, 48120, or 46689-mediated response has been elicited, e.g., by contact of the cell with 23479, 48120, or 46689 nucleic acid or protein, or administration to the cell or subject 23479, 48120, or 46689 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 23479, 48120, or 46689 (or does not express as highly as in the case of the 23479, 48120, or 46689 positive plurality of capture probes) or from a cell or subject which in which a 23479, 48120, or 46689 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding of the first sample with the binding of the second sample. Binding, e.g., in the case of a nucleic acid, hybridization with a capture probe at an address of the plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays.

[0476] In another aspect, the invention features a method of analyzing 23479, 48120, or 46689, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 23479, 48120, or 46689 nucleic acid or amino acid sequence; comparing the 23479, 48120, or 46689 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database; to thereby analyze 23479, 48120, or 46689.

[0477] Detection of Variations or Mutations

[0478] The methods of the invention can also be used to detect genetic alterations in a 23479, 48120, or 46689 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 23479, 48120, or 46689 protein activity or nucleic acid expression, such as a cellular proliferative or differentiative disorder, e.g., in the lung, brain, ovary, or breast, a neural disorder, a hematopoietic disorder, a cardiovascular disorder, or a liver disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 23479, 48120, or 46689-protein, or the mis-expression of the 23479, 48120, or 46689 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 23479, 48120, or 46689 gene; 2) an addition of one or more nucleotides to a 23479, 48120, or 46689 gene; 3) a substitution of one or more nucleotides of a 23479, 48120, or 46689 gene, 4) a chromosomal rearrangement of a 23479, 48120, or 46689 gene; 5) an alteration in the level of a messenger RNA transcript of a 23479, 48120, or 46689 gene, 6) aberrant modification of a 23479, 48120, or 46689 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a 23479, 48120, or 46689 gene, 8) a non-wild type level of a 23479, 48120, or 46689-protein, 9) allelic loss of a 23479, 48120, or 46689 gene, and 10) inappropriate post-translational modification of a 23479, 48120, or 46689-protein.

[0479] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 23479, 48120, or 46689-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 23479, 48120, or 46689 gene under conditions such that hybridization and amplification of the 23479, 48120, or 46689-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.

[0480] In another embodiment, mutations in a 23479, 48120, or 46689 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g., by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0481] In other embodiments, genetic mutations in 23479, 48120, or 46689 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 23479, 48120, or 46689 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 23479, 48120, or 46689 nucleic acid (e.g., a destabilizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 23479, 48120, or 46689 can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[0482] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 23479, 48120, or 46689 gene and detect mutations by comparing the sequence of the sample 23479, 48120, or 46689 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry.

[0483] Other methods for detecting mutations in the 23479, 48120, or 46689 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[0484] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in 23479, 48120, or 46689 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Pat. No. 5,459,039).

[0485] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 23479, 48120, or 46689 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 23479, 48120, or 46689 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0486] In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[0487] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site of the sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[0488] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0489] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 23479, 48120 or 46689 nucleic acid.

[0490] In a preferred embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or the complement of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.

[0491] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 23479, 48120, or 46689. In a preferred embodiment, each oligonucleotide of the set has a different nucleotide at an interrogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[0492] In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position. The interrogation position can be a SNP or the site of a mutation. In another preferred embodiment, the oligonucleotides of the plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides of the set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the T_(m) of the oligonucleotide. In another embodiment, at least one oligonucleotide of the set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oligonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[0493] In a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 23479, 48120, or 46689 nucleic acid.

[0494] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 23479, 48120, or 46689 gene.

[0495] Use of 23479, 48120, or 46689 Molecules as Surrogate Markers

[0496] The 23479, 48120, or 46689 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the 23479, 48120, or 46689 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 23479, 48120, or 46689 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[0497] The 23479, 48120, or 46689 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 23479, 48120, or 46689 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-23479, 48120, or 46689 antibodies may be employed in an immune-based detection system for a 23479, 48120, or 46689 protein marker, or 23479, 48120, or 46689-specific radiolabeled probes may be used to detect a 23479, 48120, or 46689 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S 16-S20.

[0498] The 23479, 48120, or 46689 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker that correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J Cancer 35:1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 23479, 48120, or 46689 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 23479, 48120, or 46689 DNA may correlate 23479, 48120, or 46689 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[0499] Pharmaceutical Compositions

[0500] The nucleic acid and polypeptides, fragments thereof, as well as anti-23479, 48120, or 46689 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0501] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0502] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0503] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0504] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0505] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0506] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0507] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0508] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0509] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0510] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0511] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0512] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0513] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[0514] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0515] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0516] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1 065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g, vincristine, vinblastine, taxol and maytansinoids). Radioactive ions include, but are not limited to iodine, yttrium and praseodymium.

[0517] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[0518] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0519] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0520] Methods of Treatment

[0521] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 23479, 48120, or 46689 expression or activity. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[0522] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 23479, 48120, or 46689 molecules of the present invention or 23479, 48120, or 46689 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0523] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 23479, 48120, or 46689 expression or activity, by administering to the subject a 23479, 48120, or 46689 or an agent which modulates 23479, 48120, or 46689 expression or at least one 23479, 48120, or 46689 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 23479, 48120, or 46689 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the 23479, 48120, or 46689 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 23479, 48120, or 46689 aberrance, for example, a 23479, 48120, or 46689, 23479, 48120, or 46689 agonist or 23479, 48120, or 46689 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[0524] It is possible that some 23479, 48120, or 46689 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[0525] The 23479, 48120, and 46689 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative or differentiative disorders, e.g., in the lung, brain, ovary, or breast, neural disorders, hematopoietic disorders, cardiovascular disorders, or liver disorders, as discussed above. The 23479, 48120, and 46689 molecules can also act as novel diagnostic targets and therapeutic agents for controlling one or more disorders associated with bone metabolism, viral diseases, and pain or metabolic disorders.

[0526] Aberrant expression and/or activity of 23479, 48120, or 46689 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 23479, 48120, or 46689 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 23479, 48120, or 46689 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 23479, 48120, or 46689 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

[0527] The 23479, 48120, or 46689 molecules of the invention may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 23479, 48120, or 46689 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 23479, 48120, or 46689 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

[0528] Additionally, 23479, 48120, or 46689 may play an important role in the regulation of metabolism or pain disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[0529] As discussed, successful treatment of 23479, 48120, or 46689 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 23479, 48120, or 46689 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)₂ and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[0530] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[0531] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[0532] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 23479, 48120, or 46689 expression is through the use of aptamer molecules specific for 23479, 48120, or 46689 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 23479, 48120, or 46689 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[0533] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 23479, 48120, or 46689 disorders. For a description of antibodies, see the Antibody section above.

[0534] In circumstances wherein injection of an animal or a human subject with a 23479, 48120, or 46689 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 23479, 48120, or 46689 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 23479, 48120, or 46689 protein. Vaccines directed to a disease characterized by 23479, 48120, or 46689 expression may also be generated in this fashion.

[0535] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0536] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 23479, 48120, or 46689 disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[0537] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0538] Another example of determination of effective dose for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate 23479, 48120, or 46689 activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of 23479, 48120, or 46689 can be readily monitored and used in calculations of IC₅₀.

[0539] Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC₅₀. An rudimentary example of such a “biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

[0540] Another aspect of the invention pertains to methods of modulating 23479, 48120, or 46689 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a 23479, 48120, or 46689 or agent that modulates one or more of the activities of 23479, 48120, or 46689 protein activity associated with the cell. An agent that modulates 23479, 48120, or 46689 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a 23479, 48120, or 46689 protein (e.g., a 23479, 48120, or 46689 substrate or receptor), a 23479, 48120, or 46689 antibody, a 23479, 48120, or 46689 agonist or antagonist, a peptidomimetic of a 23479, 48120, or 46689 agonist or antagonist, or other small molecule.

[0541] In one embodiment, the agent stimulates one or 23479, 48120, or 46689 activities. Examples of such stimulatory agents include active 23479, 48120, or 46689 protein and a nucleic acid molecule encoding 23479, 48120, or 46689. In another embodiment, the agent inhibits one or more 23479, 48120, or 46689 activities. Examples of such inhibitory agents include antisense 23479, 48120, or 46689 nucleic acid molecules, anti-23479, 48120, or 46689 antibodies, and 23479, 48120, or 46689 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 23479, 48120, or 46689 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 23479, 48120, or 46689 expression or activity. In another embodiment, the method involves administering a 23479, 48120, or 46689 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 23479, 48120, or 46689 expression or activity.

[0542] Stimulation of 23479, 48120, or 46689 activity is desirable in situations in which 23479, 48120, or 46689 is abnormally downregulated and/or in which increased 23479, 48120, or 46689 activity is likely to have a beneficial effect. For example, stimulation of 23479, 48120, or 46689 activity is desirable in situations in which a 23479, 48120, or 46689 is downregulated and/or in which increased 23479, 48120, or 46689 activity is likely to have a beneficial effect. Likewise, inhibition of 23479, 48120, or 46689 activity is desirable in situations in which 23479, 48120, or 46689 is abnormally upregulated and/or in which decreased 23479, 48120, or 46689 activity is likely to have a beneficial effect.

[0543] Pharmacogenomics

[0544] The 23479, 48120, or 46689 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 23479, 48120, or 46689 activity (e.g., 23479, 48120, or 46689 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 23479, 48120, or 46689 associated disorders (e.g., cellular proliferative or differentiative disorders, e.g., in the lung, brain, ovary, or breast, neural disorders, hematopoietic disorders, cardiovascular disorders, or liver disorders) associated with aberrant or unwanted 23479, 48120, or 46689 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 23479, 48120, or 46689 molecule or 23479, 48120, or 46689 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 23479, 48120, or 46689 molecule or 23479, 48120, or 46689 modulator.

[0545] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp, Pharmacol. Physiol. 23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0546] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase 11/1I drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[0547] Alternatively, a method termed the “candidate gene approach,” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a 23479, 48120, or 46689 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[0548] Alternatively, a method termed the “gene expression profiling,” can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 23479, 48120, or 46689 molecule or 23479, 48120, or 46689 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[0549] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 23479, 48120, or 46689 molecule or 23479, 48120, or 46689 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[0550] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more of the 23479, 48120, or 46689 genes of the present invention, wherein these products may be associated with resistance of the cells to a therapeutic agent. Specifically, the activity of the proteins encoded by the 23479, 48120, or 46689 genes of the present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more of the resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.

[0551] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a 23479, 48120, or 46689 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 23479, 48120, or 46689 gene expression, protein levels, or upregulate 23479, 48120, or 46689 activity, can be monitored in clinical trials of subjects exhibiting decreased 23479, 48120, or 46689 gene expression, protein levels, or downregulated 23479, 48120, or 46689 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 23479, 48120, or 46689 gene expression, protein levels, or downregulate 23479, 48120, or 46689 activity, can be monitored in clinical trials of subjects exhibiting increased 23479, 48120, or 46689 gene expression, protein levels, or upregulated 23479, 48120, or 46689 activity. In such clinical trials, the expression or activity of a 23479, 48120, or 46689 gene, and preferably, other genes that have been implicated in, for example, a 23479, 48120, or 46689-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[0552] 23479, 48120, or 46689 Informatics

[0553] The sequence of a 23479, 48120, or 46689 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 23479, 48120, or 46689. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination of the manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 23479, 48120, or 46689 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical, chemical or mechanical information storage device.

[0554] As used herein, “machine-readable media” refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[0555] A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

[0556] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) of the table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality of the sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymorphisms (e.g., SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[0557] By providing the nucleotide or amino acid sequences of the invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein.

[0558] Thus, in one aspect, the invention features a method of analyzing 23479, 48120, or 46689, e.g., analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 23479, 48120, or 46689 nucleic acid or amino acid sequence; comparing the 23479, 48120, or 46689 sequence with a second sequence, e.g., one or more preferably a plurality of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 23479, 48120, or 46689. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[0559] The method can include evaluating the sequence identity between a 23479, 48120, or 46689 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[0560] As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

[0561] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).

[0562] Thus, the invention features a method of making a computer readable record of a sequence of a 23479, 48120, or 46689 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[0563] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 23479, 48120, or 46689 sequence, or record, in machine-readable form; comparing a second sequence to the 23479, 48120, or 46689 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., determining if the 23479, 48120, or 46689 sequence includes a sequence being compared. In a preferred embodiment the 23479, 48120, or 46689 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. E.g., the 23479, 48120, or 46689 or second sequence can be stored in a public or proprietary database in one computer, and the results of the comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more of the following: identification of an ORF; identification of a domain, region, or site; identification of the start of transcription; identification of the transcription terminator; the full length amino acid sequence of the protein, or a mature form thereof; the 5′ end of the translated region.

[0564] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 23479, 48120, or 46689-associated disease or disorder or a pre-disposition to a 23479, 48120, or 46689-associated disease or disorder, wherein the method comprises the steps of determining 23479, 48120, or 46689 sequence information associated with the subject and based on the 23479, 48120, or 46689 sequence information, determining whether the subject has a 23479, 48120, or 46689-associated disease or disorder or a pre-disposition to a 23479, 48120, or 46689-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[0565] The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 23479, 48120, or 46689-associated disease or disorder or a pre-disposition to a disease associated with a 23479, 48120, or 46689 wherein the method comprises the steps of determining 23479, 48120, or 46689 sequence information associated with the subject, and based on the 23479, 48120, or 46689 sequence information, determining whether the subject has a 23479, 48120, or 46689-associated disease or disorder or a pre-disposition to a 23479, 48120, or 46689-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 23479, 48120, or 46689 sequence of the subject to the 23479, 48120, or 46689 sequences in the database to thereby determine whether the subject as a 23479, 48120, or 46689-associated disease or disorder, or a pre-disposition for such.

[0566] The present invention also provides in a network, a method for determining whether a subject has a 23479, 48120, or 46689 associated disease or disorder or a pre-disposition to a 23479, 48120, or 46689-associated disease or disorder associated with 23479, 48120, or 46689, said method comprising the steps of receiving 23479, 48120, or 46689 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 23479, 48120, or 46689 and/or corresponding to a 23479, 48120, or 46689-associated disease or disorder (e.g., a cellular proliferative or differentiative disorder, e.g., in the lung, brain, ovary, or breast, a neural disorder, a hematopoietic disorder, a cardiovascular disorder, or a liver disorder), and based on one or more of the phenotypic information, the 23479, 48120, or 46689 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 23479, 48120, or 46689-associated disease or disorder or a pre-disposition to a 23479, 48120, or 46689-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0567] The present invention also provides a method for determining whether a subject has a 23479, 48120, or 46689-associated disease or disorder or a pre-disposition to a 23479, 48120, or 46689-associated disease or disorder, said method comprising the steps of receiving information related to 23479, 48120, or 46689 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 23479, 48120, or 46689 and/or related to a 23479, 48120, or 46689-associated disease or disorder, and based on one or more of the phenotypic information, the 23479, 48120, or 46689 information, and the acquired information, determining whether the subject has a 23479, 48120, or 46689-associated disease or disorder or a pre-disposition to a 23479, 48120, or 46689-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0568] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

EXAMPLES Example 1

[0569] Identification and Characterization of Human 23479, 48120, or 46689 cDNA

[0570] The human 23479 nucleic acid sequence is recited as follows: TGCTGTCGCTAGATTCAGATGATTCAAGTGAGGATCAAGTGGAAAATAGTAAAAAT TCCTGGAGTTGCAAGTTTGTTGCTGCTGGAGGGCTTCAACAGTTATTAGAAATTTTT AATTCTGGAATTCTAGAGCCTAAAGAGCAGGAATCATGGACTGTGTGGCAGCTAGA CTGTCTTGCTTGCTTGCTGAAGTTAATATGCCAGTTTGCAGTAGATCCATCCGATTT GGATTTAGCTTATCATGATGTCTTTGCCTGGTCTGGTATAGCGGAAAGCCATAGGA AAAGAACCTGGCCTGGCAAATCAAGGAAGGCTGCTGGTGATCATGCTAAGGGTCTT CATATACCACGATTAACAGAGGTATTTCTTGTTCTTGTCCAAGGAACCAGTTTGATT CAGCGACTT ATGTCTGTTGCTTATACGTATGATAATCTGGCTCCTAGAGTTTTAAAA GCTCAGTCTGATCACAGGTCTAGACATGAAGTTTCACATTATTCAATGTGGCTCTTG GTGAGTTGGGCTCATTGCTGTTCTTTAGTGAAATCTAGCCTTGCTGATAGCGATCAT TTACAAGATTGGCTAAAGAAATTGACTCTCCTTATTCCTGAGACTGCAGTTCGTCAT GAATCATGCAGTGGTCTCTATAAGTTATCCCTGTCAGGGCTGGATGGAGGAGACTC AATCAATCGTTCTTTTCTGCTATTGGCTGCCTCAACATTATTGAAATTTCTTCCTGAT GCTCAAGCACTCAAACCTATTAGGATAGATGATTATGAGGAAGAACCAATATTAAA ACCAGGATGTAAAGAGTATTTTTGGTTGTTATGCAAATTAGTTGACAACATACATA TAAAGGACGCTAGTCAGACAACGCTCCTCGACTTAGATGCCTTGGCAAGACATTTG GCTGACTGTATTCGAAGTAGGGAGATCCTTGATCATCAGGATGGTAATGTAGAAGA TGATGGGCTTACAGGACTCCTAAGGCTTGCAACAAGTGTTGTTAAACACAAACCAC CCTTTAAATTTTCAAGGGAAGGACAGGAATTTTTGAGAGATATCTTCAATCTCCTGT TTTTGTTGCCAAGTCTAAAGGACCGACAACAGCCAAAGTGCAAATCACATTCTACA AGAGCTGCCGCTTACGATTTGTTAGTAGAGATGGTAAAGGGGTCTGTTGAGAACTA CAGGCTAATACACAACTGGGTTATGGCACAACACATGCAGTCCCATGCACCTTATA AATGGGATTACTGGCCTCATGAAGATGTCCGTGCTGAATGTAGATTTGTTGGCCTTA CTAACCTTGGAGCTACTTGTTACTTAGCTTCTACTATTCAGCAACTTTATATGATAC CTGAGGCAAGACAGGCTGTCTTCACTGCCAAGTATTCAGAGGATATGAAGCACAAG ACCACTCTTCTGGAGCTTCAGAAAATGTTTACATATTTAATGGAGAGTGAATGCAA AGCATATAATCCTAGACCTTTCTGTAAAACATACACCATGGATAAGCAGCCTCTGA ATACTGGGGAACAGAAAGATATGACAGAGTTTTTTACTGATCTAATTACCAAAATC GAAGAAATGTCTCCCGAACTGAAAAATACCGTCAAAAGTTTATTTGGAGGTGTAAT TACAAACAATGTTGTATCCTTGGATTGTGAACATGTTAGTCAAACTGCTGAAGAGT TTTATACTGTGAGGTGCCAAGTGGCTGATATGAAGAACATTTATGAATCTCTTGATG AAGTTACTATAAAAGACACTTTGGAAGGTGATAACATGTATACTTGTTCTCATTGTG GGAAGAAAGTACGAGCTGAAAAAAGGGCATGTTTTAAGAAATTGCCTCGCATTTTG AGTTTCAATACTATGAGATACACATTTAATATGGTCACGATGATGAAAGAGAAAGT GAATACACACTTTTCCTTCCCATTACGTTTGGACATGACGCCCTATACAGAAGATTT TCTTATGGGAAAGAGTGAGAGGAAAGAAGGTTTTAAAGAAGTCAGTGATCATTCA AAAGACTCAGAGAGCTATGAATATGACTTGATAGGAGTGACTGTTCACACAGGAA CGGCAGATGGTGGACACTATTATAGCTTTATCAGAGATATAGTAAATCCCCATGCT TATAAAAACAATAAATGGTATCTTTTTAATGATGCTGAGGTAAAACCTTTTGATTCT GCTCAACTTGCATCTGAATGTTTTGGTGGAGAGATGACGACCAAGACCTATGATTC TGTTACAGATAAATTTATGGACTTCTCTTTTGAAAAGACACACAGTGCATATATGCT GTTTTACAAACGCATGGAACCAGAGGAAGAAAATGGCAGAGAATACAAATTTGAT GTTTCGTCAGAGTTACTAGAGTGGATTTGGCATGATAACATGCAGTTTCTTCAAGAC AAAAACATTTTTGAACATACATATTTTGGATTTATGTGGCAATTGTGTAGTTGTATT CCCAGTACATTACCAGATCCTAAAGCTGTGTCCTTAATGACAGCAAAGTTAAGCAC TTCCTTTGTCCTAGAGACATTTATTCATTCTAAAGAAAAGCCCACGATGCTTCAGTG GATTGAACTGTTGACGAAACAGTTTAATAATAGTCAGGCAGCTTGTGAGTGGTTTT TAGATCGTATGGCTGATGACGACTGGTGGCCAATGCAGATACTAATTAAGTGCCCT AATCAAATTGTGAGACAGATGTTTCAGCGTTTGTGTATCCATGTGATTCAGAGGCT GAGACCTGTGCATGCTCATCTCTATTTGCAGCCAGGAATGGAAGATGGGTCAGATG ATATGGATACCTCAGTAGAAGATATTGGTGGTCGTTCATGTGTCACTCGCTTTGTGA GAACCCTGTTATTAATTATGGAACATGGTGTAAAACCTCACAGTAAACATCTTACA GAGTATTTTGCCTTCCTTTACGAATTTGCAAAAATGGGTGAAGAAGAGAGCCAATT TTTGCTTTCATTGCAAGCTATATCTACAATGGTACATTTTTACATGGGAACAAAAGG ACCTGAAAATCCTCAAGTTGAAGTGTTATCAGAGGAAGAAGGGGAAGAAGAAGAG GAGGAAGAAGATATCCTCTCTCTGGCAGAAGAAAAATACAGGCCAGCTGCCCTTG AAAAGATGATAGCTTTAGTTGCTCTTTTGGTTGAACAGTCTCGATCAGAAAGG TGA AATGTTTCGAATTTAAAATGTTTAAAGCATGTTTGGTTTTATTATTTTTACATAATTG TTTACCACTAGTTTTTCCACTAGCTTTTTATTATATATGTTTAATTATGTAATTGTTA TTCACTAGCTTTTATTATATAAATCCTTTTAAATAATACTACTATTCATCAACTCTTG TGGCATAAGAATTTCAGTTTTTTCTACCAAACTTTTACTTCATCTATGAGTCGTGTTA GAAATAGTCATTGAAAAAATATACAGTAAAATATCTAAAAAAAAAAAAAAAGG (SEQ ID NO:1).

[0571] (SEQ ID NO.1).

[0572] The human 23479 sequence (FIG. 1; SEQ ID NO: 1) is approximately 3494 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 2805 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 1; SEQ ID NO: 3). The coding sequence encodes a 934 amino acid protein (SEQ ID NO: 2), which is recited as follows: MSVAYTYDNLAPRVLKAQSDHRSRHEVSHYSMWLLVSWAHCCSLVKSSLADSDHLQ DWLKKLTLLIPETAVRHESCSGLYKLSLSGLDGGDSINRSFLLLAASTLLKFLPDAQALK PIRIDDYEEEPILKPGCKEYFWLLCKLVDNIHIKDASQTTLLDLDALARHLADCIRSREIL DHQDGNVEDDGLTGLLRLATSVVKHKPPFKFSREGQEFLRDIFNLLFLLPSLKDRQQPK CKSHSTRAAAYDLLVEMVKGSVENYRLIHNWVMAQHMQSHAPYKWDYWPHEDVRA ECRFVGLTNLGATCYLASTIQQLYMIPEARQAVFTAKYSEDMKHKTTLLELQKMFTYL MESECKAYNPRPFCKTYTMDKQPLNTGEQKDMTEFFTDLITKIEEMSPELKNTVKSLFG GVITNNVVSLDCEHVSQTAEEFYTVRCQVADMKNIYESLDEVTIKDTLEGDNMYTCSH CGKKVRAEKRACFKKLPRILSFNTMRYTFNMVTMMKEKVNTHFSFPLRLDMTPYTED FLMGKSERKEGFKEVSDHSKDSESYEYDLIGVTVHTGTADGGHYYSFIRDIVNPHAYK NNKWYLFNDAEVKPFDSAQLASECFGGEMTTKTYDSVTDKFMDFSFEKTHSAYMLFY KRMEPEEENGREYKFDVSSELLEWIWHDNMQFLQDKNIFEHTYFGFMWQLCSCIPSTL PDPKAVSLMTAKLSTSFVLETFIHSKEKPTMLQWIELLTKQFNNSQAACEWFLDRMAD DDWWPMQILIKCPNQIVRQMFQRLCIHVIQRLRPVHAHLYLQPGMEDGSDDMDTSVE DIGGRSCVTRFVRTLLLIMEHGVKPHSKHLTEYFAFLYEFAKMGEEESQFLLSLQAIST MVHFYMGTKGPENPQVEVLSEEEGEEEEEEEDILSLAEEKYRPAALEKMIALVALLVE QSRSER (SEQ ID NO:2).

[0573] The human 48120 nucleic acid sequence is recited as follows: CCACGCGTCCGGCCTAGTCCTGAGAGGCTGGGCCGGCGGCGGCTGCGGCGGGAGA CCGGTGACCCGCGGCTGGGCGCCTCGGCC ATG ACTGCGGAGCTGCAGCAGGACGA CGCGGCCGGCGCGGCAGACGGCCACGGCTCGAGCTGCCAAATGCTGTTAAATCAA CTGAGAGAAATCACAGGCATTCAGGACCCTTCCTTTCTCCATGAAGCTCTGAAGGC CAGTAATGGTGACATTACTCAGGCAGTCAGCCTTCTCACTGATGAGAGAGTTAAGG AGCCCAGTCAAGACACTGTTGCTACAGAACCATCTGAAGTAGAGGGGAGTGCTGCC AACAAGGAAGTATTAGCAAAAGTTATAGACCTTACTCATGATAACAAAGATGATCT TCAGGCTGCCATTGCTTTGAGTCTACTGGAGTCTCCCAAAATTCAAGCTGATGGAA GAGATCTTAACAGGATGCATGAAGCAACCTCTGCAGAAACTAAACGCTCAAAGAG AAAACGCTGTGAAGTCTGGGGAGAAAACCCCAATCCCAATGACTGGAGGAGAGTT GATGGTTGGCCAGTTGGGCTGAAAAATGTTGGCAATACATGTTGGTTTAGTGCTGT TATTCAGTCTCTCTTTCAATTGCCTGAATTTCGAAGACTTGTTCTCAGTTATAGTCTG CCACAAAATGTACTTGAAAATTGTCGAAGTCATACAGAAAAGAGAAATATCATGTT TATGCAAGAGCTTCAGTATTTGTTTGCTCTAATGATGGGATCAAATAGAAAATTTGT AGACCCGTCTGCAGCCCTGGATCTATTAAAGGGAGCATTCCGATCATCTGAGGAAC AGCAGCAAGATGTGAGTGAATTCACACACAAGCTCCTGGATTGGCTAGAGGACGC ATTCCAGCTAGCTGTTAATGTTAACAGTCCCAGGAACAAATCTGAAAATCCAATGG TGCAGCTGTTCTATGGTACTTTCCTGACTGAAGGGGTTCGTGAAGGAAAACCCTTTT GTAACAATGAGACCTTCGGCCAGTATCCTCTTCAGGTAAACGGTTATCGCAACTTA GACGAGTGTTTGGAAGGGGCCATGGTGGAGGGTGATGTTGAGCTTCTTCCCTCCGA TCACTCGGTGAAGTATGGACAAGAGCGTTGGTTTACAAAGCTACCTCCAGTGTTGA CCTTTGAACTCTCAAGATTTGAGTTTAATCAGTCCCTTGGGCAGCCAGAGAAAATTC ACAATAAGCTGGAATTTCCTCAGATTATTTATATGGACAGGTACATGTACAGGAGC AAGGAGCTTATTCGAAATAAGAGAGAGTGTATTCGAAAGTTGAAGGAGGAAATAA AAATTCTGCAGCAAAAATTGGAAAGGTATGTGAAATATGGCTCAGGCCCAGCTCGG TTCCCGCTCCCGGACATGCTGAAATATGTTATTGAATTTGCTAGTACAAAACCTGCC TCAGAAAGCTGTCCACCTGAAAGTGACACACATATGACATTACCACTTTCTTCAGT GCACTGCTCGGTTTCTGACCAGACATCCAAGGAAAGTACAAGTACAGAAAGCTCTT CTCAGGATGTTGAAAGTACCTTTTCTTCTCCTGAAGATTCTTTACCCAAGTCTAAAC CACTGACATCTTCTCGGTCTTCCATGGAAATGCCTTCACAGCCAGCTCCACGAACA GTCACAGATGAGGAGATAAATTTTGTTAAGACCTGTCTTCAGAGATGGAGGAGTGA GATTGAACAAGATATACAAGATTTAAAGACTTGTATTGCAAGTACTACTCAGACTA TTGAACAGATGTACTGCGATCCTCTCCTTCGTCAGGTGCCTTATCGCTTGCATGCAG TTCTTGTTCATGAAGGACAAGCAAATGCTGGACACTATTGGGCCTATATCTATAATC AACCCCGACAGAGCTGGCTCAAGTACAATGACATCTCTGTTACTGAATCTTCCTGG GAAGAAGTTGAAAGAGATTCCTATGGAGGCCTGAGAAATGTTAGTGCTTACTGTCT GATGTACATTAATGACAAACTACCCTACTTCAATGCAGAGGCAGCCCCAACTGAAT CAGATCAAATGTCAGAAGTGGAAGCCCTATCTGTGGAACTCAAGCATTACATTCAG GAGGATAACTGGCGGTTTGAGCAGGAAGTAGAGGAGTGGGAAGAAGAGCAGTCTT GCAAAATCCCTCAAATGGAGTCCTCCACCAACTCCTCATCACAGGACTACTCTACA TCACAAGAGCCTTCAGTAGCCTCTTCTCATGGGGTTCGCTGCTTGTCGTCTGAGCAT GCTGTGATTGTAAAGGAGCAAACTGCCCAGGCTATTGCAAACACAGCCCGTGCCTA TGAGAAGAGCGGTGTAGAAGCGGCACTGAGTGAGGTTAAAGAAGCTGAACCCAAG AAGCCCATGCCCCAGGAAACAAACCTTGCAGAGCAGTCAGAACAGCCCCCAAAGG CTAATGATGCAGAGTCTACTGCCCAGCCTAATTCTGAGGTCTCTGAAGTCGAGATT CCCAGTGTGGGAAGGATTCTGGTTAGATCTGATGCAGATGGATATGATGAGGAGGT GATGCTGAGCCCTGCCATGCAAGGGGTCATCCTGGCCATAGCTAAAGCCCGTCAGA CCTTTGACCGAGATGGGTCTGAAGCAGGGCTGATTAAGGCATTCCATGAAGAATAC TCCAGGCTCTATCAGCTTGCCAAAGAGACCCCCACCTCTCACAGTGATCCTCGACTT CAGCATGTCCTTGTCTACTTTTTCCAAAATGAAGCACCCAAAAGGGTAGTAGAACG AACCCTTCTGGAACAGTTTGCAGATAAAAATCTTAGCTATGATGAAAGATCAATCA GCATTATGAAGGTGGCTCAAGCGAAACTGAAGGAAATTGGTCCAGATGACATGAA TATGGAAGAGTACAAGAAGTGGCATGAAGATTATAGTTTGTTCCGAAAAGTGTCTG TGTATCTCCTAACAGGCCTAGAACTCTATCAAAAAGGAAAGTACCAAGAGGCACTT TCCTACCTGGTATATGCCTACCAGAGCAATGCTGCCCTGCTGATGAAGGGGCCCCG CCGGGGGGTCAAAGAATCCGTGATTGCTTTATACCGAAGAAAATGCCTTCTGGAGC TGAATGCCAAAGCAGCTTCTCTTTTTGAAACAAATGATGATCACTCCGTAACTGAG GGCATTAATGTGATGAATGAACTGATCATCCCCTGCATTCACCTTATCATTAATAAT GACATTTCCAAGGATGATCTGGATGCCATTGAGGTCATGAGAAACCATTGGTGCTC TTACCTTGGGCAAGATATTGCAGAAAATCTGCAGCTGTGCCTAGGGGAGTTTCTAC CCAGACTTCTAGATCCTTCTGCAGAAATCATCGTCTTGAAAGAGCCTCCAACTATTC GACCCAATTCTCCCTATGACCTATGTAGCCGATTTGCAGCTGTCATGGAGTCAATTC AGGGAGTTTCAACTGTGACAGTGAAA TAA GCTCCCACATGTTCAAGGCCCATTCTG GTTCCTGGCTGCCTGCCTCTTGCACAGAAGTTCGTTGTCATAGTGCTCACCTTGGGA AAAGGATTAGGTGGGCACATAAGATTCCGATCAGACCCCAACCATGCTGCATGTGT AAAGAAGGATTGAAAATAAAATTGCACTTTTTAGGTACAAAATCATAAAAGCTGTT TCACTAGAAAAGGCAGAAAGCAGTGTATTAAGGTGTTGAATTACGCCAGAAGACC TGAAATGCCTTGTACCTACAACAATGCTTAGGCTTTTCTAAGCCTCTTGCCACTTTT AAAATTATCCTTCAGGCATAAATATTTTTGACAGCAGAATAGAAGAATGATTCATG AGAACCTGAACCAGATGAACAGCTACTAGTTATTTTATCAAATACAGATGACATTT AAAAATTCTTAACTACAAGAGATTAGAAATATAAACCTTGCCTGGCTCTTGCCAGG AGATAACAAAATGGGTTGCTGATGAACTGCACCCTTTTACATGTGGGTAGAATATA AGCTCACATGGCAGTGAGATGTTGAAAAGTCAAAAGAGACCTGTCTCTCTCCTTTC TTTTCTATCTTTAAACCAGAAAACCTCATACTCAGTCCTCAGTGAAAGAAAGTAAA GTATTAAGGACTTTAGGCAGAAGAGCATTGTGTAACTTGACTGAAGATCATCCATT AATAGTTATTAGGCATTTAGGTAAAATTTTCTAATACCTAAAAATTGTCAAAAACA GTCAATAGGGCTACTGCTGGCCCAAAGACCATTTAGGTCCACCTCCTCTTTTTTGCT CTTTTTTTTTTTTCTGTGACAGTTTCACTGTGTCGCCCAGGCTGGCGTTCAGTGGTGC AATCTCAGCTCACTGCAAACTCTGTCTCCTGGGCTCAAGTGATTCTCGTGCCTCAGC CTCCCGAATAGCTGGAATTACGGGCATGCACCACCACACCTGGCTAATTTTTGTATT TTTAATAGAGATGGGGTTTCACCATATTGGCCAGGCTGATCTCTAACTCCTGGCCTC AAGTGATCTATCTGCCTCCCTCAGCCTCCCAAAGTCTGGGATTGCAGACAAGTCAT CGTACCCGGCCTTCTTTTTTGCCCTTAAAAGTAAGGGATGTGGGTTTGTACAAAAAA AAAAAAAAAAAAAAAAAAAAACCAGCATACATATGCAAAACTATATATATATGTA TATGTAGAGAAAAATACTTCCCATTGATCATTTTTAAAAGGCTTCTGATTGGATATT GTGTTTTAACCAAATTTTAAAGATTAATGGAATCATGAAAGGGAAAAAATTGATAC AACTATGCAGATTTTATAAATGTGCAATAAAAGTATTTGTTTTACA (SEQ ID NO:4).

[0574] The human 48120 sequence (FIG. 3; SEQ ID NO: 4) is approximately 4873 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA) which are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 3420 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 4; SEQ ID NO: 6). The coding sequence encodes a 1139 amino acid protein (SEQ ID NO: 5), which is recited as follows: MTAELQQDDAAGAADGHGSSCQMLLNQLREITGIQDPSFLHEALKASNGDITQAVSLL TDERVKEPSQDTVATEPSEVEGSAANKEVLAKVIDLTHDNKDDLQAAIALSLLESPKIQ ADGRDLNRMHEATSAETKRSKRKRCEVWGENPNPNDWRRVDGWPVGLKNVGNTCW FSAVIQSLFQLPEFRRLVLSYSLPQNVLENCRSHTEKRNIMFMQELQYLFALMMGSNRK FVDPSAALDLLKGAFRSSEEQQQDVSEFTHKLLDWLEDAFQLAVNVNSPRNKSENPMV QLFYGTFLTEGVREGKPFCNNETFGQYPLQVNGYRNLDECLEGAMVEGDVELLPSDHS VKYGQERWFTKLPPVLTFELSRFEFNQSLGQPEKIHNKLEFPQIIYMDRYMYRSKELIRN KRIECIRKLKEEIKILQQKLERYVKYGSGPARFPLPDMLKYVIEFASTKPASESCPPESDT HMTLPLSSVHCSVSDQTSKESTSTESSSQDVESTFSSPEDSLPKSKPLTSSRSSMEMPSQP APRTVTDEEINFVKTCLQRWRSEIEQDIQDLKTCIASTTQTIEQMYCDPLLRQVPYRLHA VLVHEGQANAGHYWAYIYNQPRQSWLKYNDISVTESSWEEVERDSYGGLRNVSAYCL MYINDKLPYFNAEAAPTESDQMSEVEALSVELKHYIQEDNWRFEQEVEEWEEEQSCKI PQMESSTNSSSQDYSTSQEPSVASSHGVRCLSSEHAVIVKEQTAQAIANTARAYEKSGV EAALSEVKEAEPKKPMPQETNLAEQSEQPPKANDAESTAQPNSEVSEVEIPSVGRILVRS DADGYDEEVMLSPAMQGVILAIAKARQTFDRDGSEAGLIKAFHEEYSRLYQLAKETPT SHSDPRLQHVLVYFFQNEAPKRVVERTLLEQFADKNLSYDERSISIMKVAQAKLKEIGP DDMNMEEYKKWHEDYSLFRKVSVYLLTGLELYQKGKYQEALSYLVYAYQSNAALL MKGPRRGVKESVIALYRRKCLLELNAKAASLFETNDDHSVTEGINVMNELIIPCIHLIIN NDISKDDLDAIEVMRNHWCSYLGQDIAENLQLCLGEFLPRLLDPSAEIIVLKEPPTIRPNS PYDLCSRFAAVMESIOGVSTVTVK (SEQ ID NO:5).

[0575] The human 46689 nucleic acid sequence is recited as follows: CACTAGTAACGCCGCCATGTGCTGGAATTCGCCCTTCTCGGGAAGCGCGCCATTGT GTTGGTACCCGGGAATTCGCGGCCGCGTCGACGCCCGCCGGGGCTCTCCAGCTTCG CC ATGCCGCCGTGGGGCGCCGCCCTCGCGCTCATCTTGGCCGTGCTCGCCCTTCTCG GCCTGCTCGGCCCGCGGCTCCGGGGACCCTGGGGGCGCGCCGTCGGAGAGAGGAC CCTGCCGGGGGCCCAAGACCGAGACGACGGGGAGGAGGCGGACGGCGGAGGCCC GGCGGACCAGTTCAGCGACGGGCGCGAGCCACTGCCGGGAGGGTGCAGCCTTGTTT GCAAGCCGTCGGCCCTGGCCCAGTGCCTGCTGCGCGCCCTGCGGCGCTCAGAGGCG CTGGAGGCCGGCCCGCGCTCCTGGTTCTCCGGGCCCCACCTGCAGACCCTCTGCCA CTTCGTCCTGCCCGTAGCGCCTGGGCCTGAGCTGGCCCGGGAGTACCTGCAGTTGG CGGACGATGGGCTAGTGGCCCTGGACTGGGTGGTAGGACCTTGTGTTCGGGGCCGC CGGATCACCAGCGCCGGGGGCCTTCCTGCGGTGCTTCTGGTGATCCCCAATGCGTG GGGTCGCCTCACCCGCAACGTGCTCGGCCTTTGCTTGCTCGCCCTGGAGCGCGGCT ACTACCCGGTCATCTTCCATCGCCGCGGCCACCACGGTTGCCCACTGGTCAGCCCCC GGCTGCAGCCTTTCGGGGACCCGTCCGACCTCAAGGAGGCGGTCACATACATCCGC TTCCGACACCCGGCGGCGCCGCTGTTCGCGGTGAGCGAAGGCTCGGGCTCGGCGCT GCTCCTGTCCTACCTGGGCGAGTGCGGCTCCTCCAGCTACGTGACAGGCGCCGCCT GCATCTCGCCCGTGCTGCGCTGCCGAGAGTGGTTCGAGGCCGGCCTGCCCTGGCCC TACGAGCGGGGCTTTCTGCTCCACCAGAAGATCGCCCTCAGCAGGTATGCCACAGC CCTGGAGGACACTGTGGACACCAGCAGACTGTTCAGGAGCCGTTCCCTTCGAGAGT TTGAGGAGGCTCTCTTCTGCCACACCAAAAGCTTCCCCATCAGCTGGGATGCCTACT GGGACCGCAACGACCCGCTCCGGGATGTCGATGAGGCAGCCGTGCCTGTGCTGTGT ATCTGCAGTGCTGACGACCCCGTGTGTGGACCCCCAGACCACACTCTGACAACTGA ACTCTTCCACAGCAACCCCTACTTCTTCCTCCTGCTCAGTCGCCACGGAGGCCACTG TGGCTTCCTGCGCCAGGAGCCCTTGCCAGCCTGGAGCCATGAGGTCATCTTGGAGT CCTTCCGGGCCTTGACTGAGTTCTTCCGAACGGAGGAGAGGATTAAAGGGCTGAGC AGGCACAGAGCTTCCTTCCTTGGGGGCCGTCGTCGTGGGGGAGCCTTGCAGAGGCG GGAAGTCTCTTCCTCTTCCAACCTGGAGGAGATCTTTAACTGGAAGCGATCATACA CAAGG TGA GAGACCTGGCCTGAGAACCCCCAAGTCCTGCAAAGAAAAACAGAGCT GGGCAAGGGGGAGTCCTGGAAAGATGGGGCGGACTGAACAGAGGGAGCTCCAGCT CTGTGCTCCTCATTCAGTCCCTCTCTCTTAAATTGGTGCCTTGAAAGAGAAGGAACG TCCTGCGAGCCTGCACTCACTTCATCCTCAGCAGAACTCCTGCCTGGCCTCTGCTCA ACATATCCCTACTCATCCGGTCAGCAGCGGCGCGTTCCAGTCACTGTCACCTGTCAC TGACATCACAAGCCAAAGGATAGCACTTTTTCAATCCATGGACTCAGGAGAAAATG CCCTCTTACTGGCAGTGGCTAGAGGGATGAGACGTTTGTGTATGTCACTGGGCAGT GACCCCGATTCTCAAGCTGGAGCCATTTGATGTCATGAGGACAGGATGTTTGTGTC TCGGCCCCACTTCCCTCATTTGCTCTGTGGTTGTGGCGCCCTGCTTTGACCGAATGC TCTGGCAACTGCGGCAGCAGGCTTGTGTGTGTGAGAAGGGCGGCAGAGGCAGTGG GGCTGGCT (SEQ ID NO:7).

[0576] The human 46689 sequence (FIG. 1; SEQ ID NO: 7), which is approximately 2082 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) that are underscored above. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 1407 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO: 7; SEQ ID NO: 9). The coding sequence encodes a 468 amino acid protein (SEQ ID NO: 8), which is recited as follows: MPPWGAALALILAVLALLGLLGPRLRGPWGRAVGERTLPGAQDRDDGEEADGGGPAD QFSDGREPLPGGCSLVCKPSALAQCLLRALRRSEALEAGPRSWFSGPHLQTLCHFVLPV APGPELAREYLQLADDGLVALDWVVGPCVRGRRITSAGGLPAVLLVIPNAWGRLTRN VLGLCLLALERGYYPVIFHRRGHHGCPLVSPRLQPFGDPSDLKEAVTYIRFRHPAAPLF AVSEGSGSALLLSYLGECGSSSYVTGAACISPVLRCREWFEAGLPWPYERGFLLHQKIA LSRYATALEDTVDTSRLFRSRSLREFEEALFCHTKSFPISWDAYWDRNDPLRDVDEAAV PVLCICSADDPVCGPPDHTLTTELFHSNPYFFLLLSRHGGHCGFLRQEPLPAWSHEVILE SFRALTEFFRTEERIKGLSRHRASFLGGRRRGGALQRREVSSSSNLEEIFNWKRSYTR (SEQ ID NO:8).

Example 2:

[0577] Tissue Distribution of 23479, 48120, or 46689 mRNA by TagMan Analysis

[0578] Endogenous human 23479, 48120, or 46689 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5′ end (typically 6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount of the gene of interest in the test sample, thus providing a quantitative measure of the initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5′ end (typically VIC).

[0579] To determine the level of 23479 and 48120 in various human tissues, primer/probe sets were designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Tables 2 and 3. 23479 mRNA was detected in coronary SMC, HUVEC, normal brain cortex, brain hypothalamus, lung tumor, and erythroid cells (Table 2). 48120 expression was found in heart, heart CHF, normal brain cortex, and lung tumor (Table 3).

[0580] To determine the level of 46689 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Tables 1-7. 46689 mRNA was detected in hematopoietic cells (e.g., bone-marrow mononuclear cells), lung, thymus, glial cells, kidney, and the brain (e.g., the cortex), as well as in numerous tumors from the lung, brain, breast, and ovary. TABLE 2 Expression of 23479 mRNA Relative Tissue Expression Artery normal 136.3135 Aorta diseased 23.0355 Vein normal 6.7776 Coronary Smooth Muscle Cells (SMC) 219.1514 Human Umbilical Vein Endothelial Cells (HUVEC) 829.3195 Hemangioma 36.3979 Heart normal 102.9489 Heart Congestive Heart Failure 89.6222 Kidney 122.8526 Skeletal Muscle 170.755 Adipose normal 5.4861 Pancreas 37.6815 primary osteoblasts 44.1942 Osteoclasts (differentiated) 0 Skin normal 4.3796 Spinal cord normal 37.4212 Brain Cortex normal 675.9554 Brain Hypothalamus normal 219.9123 Nerve 32.1286 Dorsal Root Ganglion 54.7879 Breast normal 29.2585 Breast tumor 69.8304 Ovary normal 88.3883 Ovary Tumor 1.0761 Prostate Normal 6.7308 Prostate Tumor 54.7879 Salivary glands 3.2848 Colon normal 0.7689 Colon Tumor 27.3939 Lung normal 0.5929 Lung tumor 184.2837 Lung Chronic Obstructive Pulmonary Disease 1.3433 Colon Inflammatory Bowel Disease 0.2358 Liver normal 2.015 Liver fibrosis 11.2028 Spleen normal 2.7147 Tonsil normal 14.18 Lymph node normal 10.0616 Small intestine normal 3.0121 Skin-Decubitus 8.6086 Synovium 0.7026 Bone Marrow, Mononuclear Cells 6.9441 Activated peripheral blood mononuclear cells 2.7241 Neutrophils 16.9215 Megakaryocytes 89.9333 Erythroid 424.8425

[0581] Expression of human 23479 mRNA was detected in many tissues. Prominent expression was detected in cardiovascular tissues (e.g., arteries, smooth muscle cells, endothelial cells, and the heart), kidney, skeletal muscle, brain (e.g., the cortex and hypothalamus), ovary, and blood cells (e.g., megakaryocytes and erythroid cells). Significantly, expression of human 23479 was elevated in lung, breast, prostate, and colon tumors, as compared to its expression in the appropriate normal tissues. In contrast, expression of human 23479 was decreased in ovary tumors as compared to its expression in normal ovary tissue. TABLE 3 Expression of 48120 mRNA Relative Tissue Expression Artery normal 5.9826 Aorta diseased 0.6858 Vein normal 0.398 Coronary Smooth Muscle Cells 8.6986 Human Umbilical Vein Endothelial Cells 13.9848 Hemangioma 2.3388 Heart normal 34.5541 Heart congestive heart failure 104.0248 Kidney 3.9334 Adipose normal 0 Pancreas 3.3422 primary osteoblasts 0.2025 Osteoclasts (differentiated) 0 Skin normal 0 Spinal cord normal 0.4832 Brain Cortex normal 39.83 Brain Hypothalamus normal 6.8485 Nerve 1.8542 Dorsal Root Ganglion 1.348 Breast normal 1.6142 Breast tumor 1.249 Ovary normal 5.962 Ovary Tumor 0.4832 Prostate Normal 1.9531 Prostate Tumor 4.4716 Salivary glands 0.231 Colon normal 0.3133 Colon Tumor 5.2082 Lung normal 0.1567 Lung tumor 49.8936 Lung Chronic Obstructive Pulmonary Disease 0.2247 Colon Inflammatory Bowel Disease 0.06 Liver normal 0.3898 Liver fibrosis 1.334 Spleen normal 1.5592 Tonsil normal 1.8931 Lymph node normal 1.6367 Small intestine normal 0.4325 Macrophages 0.0183 Synovium 0 Bone Marrow, Mononuclear Cells 0.0363 Activated peripheral blood mononuclear cells 0.0754 Neutrophils 0.5628 Megakaryocytes 0.0407 Erythroid 0.5325 positive control 59.1286 Skeletal Muscle 21.8685

[0582] Expression of human 48120 mRNA was detected in many tissues, including cardiovascular tissues (e.g., arteries, smooth muscle cells, endothelial cells, and the heart), kidney, pancreas, skeletal muscle, brain (e.g., the cortex and hypothalamus), breast, ovary, prostate, spleen, tonsil, and lymph node tissue. Significantly, expression of human 48120 was highly elevated in heart tissue from a patient that suffered from congestive heart failure. In addition, expression of human 48120 was elevated in lung, colon and prostate tumors, as compared to its expression in the appropriate normal tissues. In contrast, expression of human 48120 was decreased in ovary tumors as compared to its expression in normal ovary tissue. TABLE 4 Expression of 46689 mRNA Relative Tissue Expression Artery normal 0 Vein normal 0 Aortic SMC EARLY 6.23 Aortic SMC LATE 5.24 Static HUVEC 7.13 Shear HUVEC 7.88 Heart normal 0.94 Heart CHF 1.33 Kidney 10.4 Skeletal Muscle 1.19 Adipose normal 7.46 Pancreas 4.67 Primary Osteoblasts 2 Osteoclasts (diff) 0.16 Skin normal 2.69 Spinal cord normal 1.18 Brain Cortex normal 8.07 Brain Hypothalamus normal 3.97 Nerve 3.44 DRG (Dorsal Root Ganglion) 3.31 Glial Cells (Astrocytes) 12.8 Glioblastoma 2.65 Breast normal 6.51 Breast tumor 104.57 Ovary normal 0.23 Ovary Tumor 8.84 Prostate Normal 5.04 Prostate Tumor 0.47 Epithelial Cells (Prostate) 0.36 Colon normal 0.09 Colon Tumor 2.83 Lung normal 40.18 Lung tumor 16.89 Lung COPD 81.76 Colon JBD 2.45 Liver normal 2.26 Liver fibrosis 4.01 Dermal Cells- fibroblasts 6.25 Spleen normal 6.4 Tonsil normal 3.68 Lymph node 0.03 Thymus normal 22.6 Skin-Decubitus 9.57 Synovium (rheumatoid Arthritis) 49.63 BM-MNC 47.94 Activated PBMC 0.63

[0583] Expression of human 46689 mRNA was detected in bone marrow mononuclear cells (BM-MNC), lung, thymus, glial cells, kidney, brain cortex, human umbilical vein endothelial cells (HUVECs), aortic smooth muscle cells (SMCs), heart, skeletal muscle, adipose tissue, pancreas, osteoblasts and osteoclasts, skin, spinal chord, hypothalamus, dorsal root ganglia (DRG) and other nerve cells, breast, ovary, prostate, prostate epithelial cells, colon, liver, dermal fibroblasts, spleen, tonsil, lymph nodes, and activated pre bone marrow cells. In addition, human 46689 is expressed in tissues, cells, or fluids associated with disease, including a breast tumor, lung tissue from a patient with chronic obstructive pulmonary disease, sinovium from a patient with rheumatoid arthritis, a lung tumor, decubitus skin, an ovary tumor, liver tissue from a patient with liver fibrosis, a colon tumor, colon tissue form a patient with inflammatory bowel disease (IBD), a prostate tumor, and heart tissue from a patient that had congestive heart failure. Importantly, 46689 expression was elevated in the breast, ovary, and colon tumors as compared to the appropriate normal tissue. TABLE 5 Expression of 46689 mRNA Relative Tissue Expression Hemangioma 0.0 Hemangioma 0.9 Hemangioma 0.5 Normal Kidney 4.8 Renal Cell Carcinoma 0.3 Wilms Tumor 6.4 Wilms Tumor 4.2 Skin 0.0 Uterine Adenocarcinoma 1.2 Neuroblastoma 1.2 Fetal Adrenal 0.4 Fetal Kidney 4.8 Fetal Heart 1.4 Normal Heart 2.1 Cartilage 1.9 Spinal cord 4.2 lymphangioma 2.8 Endometrial polyps 0.0 Synovium (RA) 0.4 Hyperkeratotic skin 1.8

[0584] Expression of 46689 was detected in normal kidney, fetal kidney, fetal adrenal gland, normal heart, fetal heart, cartilage, and spinal chord tissues. In addition, expression of 46689 was observed in several samples associated with disease, including Wilms tumors, a lymphangioma, hyoperkaratotic skin, a uterine adenocarcinoma, a neuroblastoma, hemangiomas, synovial fluid from a patient with rheumatoid arthritis (RA), and a renal cell carcinoma. TABLE 6 Expression of 46689 mRNA Relative Tissue Expression PIT 400 Breast N 0.49 PIT 56 Breast N 7.34 MDA 106 Breast T 1.17 MDA 234 Breast T 0.46 NDR 57 Breast T 1.02 MDA 304 Breast T 0.85 NDR 58 BreastT 2.84 NDR 132 Breast T 6.99 NDR 07 Breast T 0.33 NDR 12 BreastT 13.00 PIT 208 Ovary N 1.81 CHT 620 Ovary N 1.05 CHT 619 Ovary N 1.79 CLN 03 Ovary T 1.24 CLN 05 Ovary T 3.57 CLN 17 Ovary T 2.78 CLN 07 Ovary T 0.43 CLN 08 Ovary T 0.30 MDA 216 Ovary T 0.52 CLN 012 Ovary T 4.63 MDA 25 Ovary T 5.86 MDA 183 Lung N 0.08 CLN 930 Lung N 0.59 MDA 185 Lung N 0.51 CHT 816 Lung N 0.21 MPI 215 Lung T--SmC 3.51 MDA 259 Lung T-PDNSCCL 2.14 CHT 832 Lung T-PDNSCCL 5.05 MDA 253 Lung T-PDNSCCL 3.96 CHT 814 Lung T-SCC 5.90 CHT 911 Lung T-SCC 16.01 CHT 726 Lung T-SCC 0.91 CHT 845 Lung T-AC 11.01

[0585] Expression of human 46689 was detected in normal breast, ovary, and lung tissue samples, as well as in breast, ovary, and lung tumors. Significantly, expression of human 46689 mRNA was elevated in 8/8 lung tumors analyzed, 4/7 ovary tumors analyzed, and 1/8 breast tumors analyzed, as compared to normal lung, ovary, and breast tissue samples, respectively. This indicates that human 46689 is a marker of tumor formation and/or growth, especially in the lung. Abbreviation used: N, normal; T, tumor; SmC, small cell carcinoma; PDNSCCL, poorly differentiated non-small cell carcinoma of the lung; SCC, squamous cell carcinoma; AC, adenocarcinoma. TABLE 7 Expression of 46689 mRNA Relative Tissue Expression Colon N 0.33 Colon N 4.51 Colon N 2.66 Colon N 23.63 Colon T 0.30 Colon T 0.52 Colon T 6.18 Colon T 1.88 Colon T 0.20 Liver Met 0.20 Liver Met 0.14 Liver Met 0.32 Liver Met 0.18 Liver Nor 0.10 Liver Nor 0.27 Brain N 0.04 Brain N 0.05 Brain N 0.11 Brain N 0.05 Astrocytes 0.74 Brain T 1.85 Brain T 0.91 Brain T 0.35 Brain T 0.30 HMVEC-Arrested 0.13 HMVEC-Proliferating 0.17 Placenta 0.49 Fetal Adrenal 0.14 Fetal Adrenal 0.00 Fetal Liver 0.11 Fetal Liver 2.85 Wilms T 0.07 Renal T 0.00 Endometrial AC 0.13

[0586] Expression of 46689 mRNA was detected in normal colon, liver, and brain tissue, as well as in human vascular endothelial cells (HMVEC), fetal adrenal gland, and fetal liver. Expression of 46689 was also detected in colon tumors, liver metastases, brain tumors, a wilms tumor, a renal tumor, and an endometrial adenocarcinoma (AC). Significantly, 4/4 brain tumors analyzed displayed elevated 46689 mRNA expression as compared to normal brain tissue, again suggesting that human 46689 is a marker for tumor formation and/or growth in some tissue. Abbreviations used: N, normal; T, tumor; Met, metastasis. TABLE 8 Expression of 46689 mRNA Relative Tissue Expression PIT 337 Colon N 13.09 CHT 410 Colon N 4.32 CUT 425 Colon N 4.46 CHT 371 Colon N 4.02 PIT 281 Colon N 7.21 NDR 211 Colon N 4.50 CHT 122 Adenomas 6.50 CUT 887 Adenomas 21.64 CHT 414 Colonic ACA-B 9.29 CUT 841 Colonic ACA-B 8.00 CUT 890 Colonic ACA-B 3.09 CUT 910 Colonic ACA-B 3.34 CUT 807 Colonic ACA-B 3.75 CUT 382 Colonic ACA-B 3.92 CUT 377 Colonic ACA-B 3.04 CUT 520 Colonic ACA-C 4.58 CUT 596 Colonic ACA-C 4.04 CUT 907 Colonic ACA-C 6.97 CHT 372 Colonic ACA-C 11.05 NDR 210 Colonic ACA-C 9.26 CHT 1365 Colonic ACA-C 6.75 CLN 740 Liver N 26.83 CLN 741 Liver N 26.64 NDR 165 Liver N 6.11 NDR 150 Liver N 20.05 PIT 236 Liver N 5.68 CHT 1878 Liver N 21.27 CHT 077 Colon to Liver Met 7.73 CHT 119 Colon to Liver Met 21.64 CHT 131 Colon to Liver Met 18.07 CHT 218 Colon to Liver Met 14.73 CHT 739 Colon to Liver Met 14.63 CHT 755 Colon to Liver Met 18.52 CHT 215 Col Abdominal Met 2.57

[0587] Expression of 46689 mRNA was detected in both normal colon and liver tissues, as well as in colon tumors and liver metastases. Of the colon tumors, 1/15 displayed an increase in 46689 expression relative to normal colon tissue. Abbreviations used: N, normal; ACA, adenocarcinoma; Met, metastasis. TABLE 9 Expression of 46689 mRNA Relative Cell line Expression MCF-7 Breast T 53.47 ZR75 Breast T 44.66 T47D Breast T 49.38 MDA 231 Breast T 19.71 MDA 435 Breast T 7.81 SKBr3 Breast 19.78 DLD 1 ColonT (stageC) 113.83 HCT116 Colon T 31.58 HT29 Colon T 42.25 Cob 205 Colon T 15.68 NCIH125 Lung T 8.34 A549 Lung T 55.36 NHBE Lung 33.49 SKOV-3 ovary 94.08 OVCAR-3 ovary 10.82 293 Baby Kidney 6.71 293T Baby Kidney 19.92

[0588] Expression of 46689 mRNA in cell lines that are suitable for transplantation into mice as part of a xenografting experiment. All cell lines displayed high expression of human 46689, with the exception of MDA 435 Breast tumor cells, NCIH 125 Lung tumor cells, and 293 Baby Kidney cells. Expression of 46689 mRNA is particularly high in stage 3 DLD1 Colon tumor cells and SKOV-3 Ovary cells. TABLE 10 Expression of 46689 mRNA Relative Tissue Expression RIP Angio 9.0366 RIP Tumor 13.9848 Xeno Parent 1 3.6955 Xeno Parent 2 0.0566 Xeno VEGF 1 0.1763 Xeno VEGF 2 0.251 Spleen 3.0754 Heart 0.4078 Kidney 0.2536 Liver 3.3422 VEGF 1 0.862 VEGF 2 1.3294 P1 0.4431 P2 0.2853

[0589] Cells expressing human 46689 were transplanted into mice and allowed to form tumors in vivo. The tumor cells then isolated and analyzed for 46689 expression. RIP Angio shows 46689 expression in cells that were removed just after angiogenesis had begun to provide blood vessels to the tumor, while RIP Tumor shows 46689 expression in cells that were removed after angiogenesis had contributed to further tumor growth. Angiogenesis-dependent tumor growth was correlated with an increase in 46689 expression. The Xeno VEGF 1, Xeno VEGF2, VEGF1, and VEGF2 values correspond to 46689 expression levels for cells transplanted into mice that overexpress either VEGF1 or VEGF2, growth factors involved in angiogenesis. The Xeno Parent 1, Xeno Parent 2, P1, and P2 lanes are control mice that do not overexpress either VEGF 1 or VEGF2.

Example 3

[0590] Tissue Distribution of 23479, 48120, or 46689 mRNA by Northern Analysis

[0591] Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the 23479, 48120, or 46689 cDNA (SEQ ID NO: 1) can be used. The DNA was radioactively labeled with ³²P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 4

[0592] Recombinant Expression of 23479, 48120, or 46689 in Bacterial Cells

[0593] In this example, 23479, 48120, or 46689 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 23479, 48120, or 46689 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-23479, 48120, or 46689 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB 199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 5

[0594] Expression of Recombinant 23479, 48120, or 46689 Protein in COS Cells

[0595] To express the 23479, 48120, or 46689 gene in COS cells (e.g., COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) CellI23:175-182), the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 23479, 48120, or 46689 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[0596] To construct the plasmid, the 23479, 48120, or 46689 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the 23479, 48120, or 46689 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the 23479, 48120, or 46689 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the 23479, 48120, or 46689gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB 101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[0597] COS cells are subsequently transfected with the 23479, 48120, or 46689-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The expression of the 23479, 48120, or 46689 polypeptide is detected by radiolabelling (³⁵S-methionine or ³⁵5-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[0598] Alternatively, DNA containing the 23479, 48120, or 46689 coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the 23479, 48120, or 46689 polypeptide is detected by radiolabelling and immunoprecipitation using a 23479, 48120, or 46689 specific monoclonal antibody.

[0599] Equivalents

[0600] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

1 20 1 3494 DNA Homo sapiens CDS (405)...(3206) 1 tgctgtcgct agattcagat gattcaagtg aggatcaagt ggaaaatagt aaaaattcct 60 ggagttgcaa gtttgttgct gctggagggc ttcaacagtt attagaaatt tttaattctg 120 gaattctaga gcctaaagag caggaatcat ggactgtgtg gcagctagac tgtcttgctt 180 gcttgctgaa gttaatatgc cagtttgcag tagatccatc cgatttggat ttagcttatc 240 atgatgtctt tgcctggtct ggtatagcgg aaagccatag gaaaagaacc tggcctggca 300 aatcaaggaa ggctgctggt gatcatgcta agggtcttca tataccacga ttaacagagg 360 tatttcttgt tcttgtccaa ggaaccagtt tgattcagcg actt atg tct gtt gct 416 Met Ser Val Ala 1 tat acg tat gat aat ctg gct cct aga gtt tta aaa gct cag tct gat 464 Tyr Thr Tyr Asp Asn Leu Ala Pro Arg Val Leu Lys Ala Gln Ser Asp 5 10 15 20 cac agg tct aga cat gaa gtt tca cat tat tca atg tgg ctc ttg gtg 512 His Arg Ser Arg His Glu Val Ser His Tyr Ser Met Trp Leu Leu Val 25 30 35 agt tgg gct cat tgc tgt tct tta gtg aaa tct agc ctt gct gat agc 560 Ser Trp Ala His Cys Cys Ser Leu Val Lys Ser Ser Leu Ala Asp Ser 40 45 50 gat cat tta caa gat tgg cta aag aaa ttg act ctc ctt att cct gag 608 Asp His Leu Gln Asp Trp Leu Lys Lys Leu Thr Leu Leu Ile Pro Glu 55 60 65 act gca gtt cgt cat gaa tca tgc agt ggt ctc tat aag tta tcc ctg 656 Thr Ala Val Arg His Glu Ser Cys Ser Gly Leu Tyr Lys Leu Ser Leu 70 75 80 tca ggg ctg gat gga gga gac tca atc aat cgt tct ttt ctg cta ttg 704 Ser Gly Leu Asp Gly Gly Asp Ser Ile Asn Arg Ser Phe Leu Leu Leu 85 90 95 100 gct gcc tca aca tta ttg aaa ttt ctt cct gat gct caa gca ctc aaa 752 Ala Ala Ser Thr Leu Leu Lys Phe Leu Pro Asp Ala Gln Ala Leu Lys 105 110 115 cct att agg ata gat gat tat gag gaa gaa cca ata tta aaa cca gga 800 Pro Ile Arg Ile Asp Asp Tyr Glu Glu Glu Pro Ile Leu Lys Pro Gly 120 125 130 tgt aaa gag tat ttt tgg ttg tta tgc aaa tta gtt gac aac ata cat 848 Cys Lys Glu Tyr Phe Trp Leu Leu Cys Lys Leu Val Asp Asn Ile His 135 140 145 ata aag gac gct agt cag aca acg ctc ctc gac tta gat gcc ttg gca 896 Ile Lys Asp Ala Ser Gln Thr Thr Leu Leu Asp Leu Asp Ala Leu Ala 150 155 160 aga cat ttg gct gac tgt att cga agt agg gag atc ctt gat cat cag 944 Arg His Leu Ala Asp Cys Ile Arg Ser Arg Glu Ile Leu Asp His Gln 165 170 175 180 gat ggt aat gta gaa gat gat ggg ctt aca gga ctc cta agg ctt gca 992 Asp Gly Asn Val Glu Asp Asp Gly Leu Thr Gly Leu Leu Arg Leu Ala 185 190 195 aca agt gtt gtt aaa cac aaa cca ccc ttt aaa ttt tca agg gaa gga 1040 Thr Ser Val Val Lys His Lys Pro Pro Phe Lys Phe Ser Arg Glu Gly 200 205 210 cag gaa ttt ttg aga gat atc ttc aat ctc ctg ttt ttg ttg cca agt 1088 Gln Glu Phe Leu Arg Asp Ile Phe Asn Leu Leu Phe Leu Leu Pro Ser 215 220 225 cta aag gac cga caa cag cca aag tgc aaa tca cat tct aca aga gct 1136 Leu Lys Asp Arg Gln Gln Pro Lys Cys Lys Ser His Ser Thr Arg Ala 230 235 240 gcc gct tac gat ttg tta gta gag atg gta aag ggg tct gtt gag aac 1184 Ala Ala Tyr Asp Leu Leu Val Glu Met Val Lys Gly Ser Val Glu Asn 245 250 255 260 tac agg cta ata cac aac tgg gtt atg gca caa cac atg cag tcc cat 1232 Tyr Arg Leu Ile His Asn Trp Val Met Ala Gln His Met Gln Ser His 265 270 275 gca cct tat aaa tgg gat tac tgg cct cat gaa gat gtc cgt gct gaa 1280 Ala Pro Tyr Lys Trp Asp Tyr Trp Pro His Glu Asp Val Arg Ala Glu 280 285 290 tgt aga ttt gtt ggc ctt act aac ctt gga gct act tgt tac tta gct 1328 Cys Arg Phe Val Gly Leu Thr Asn Leu Gly Ala Thr Cys Tyr Leu Ala 295 300 305 tct act att cag caa ctt tat atg ata cct gag gca aga cag gct gtc 1376 Ser Thr Ile Gln Gln Leu Tyr Met Ile Pro Glu Ala Arg Gln Ala Val 310 315 320 ttc act gcc aag tat tca gag gat atg aag cac aag acc act ctt ctg 1424 Phe Thr Ala Lys Tyr Ser Glu Asp Met Lys His Lys Thr Thr Leu Leu 325 330 335 340 gag ctt cag aaa atg ttt aca tat tta atg gag agt gaa tgc aaa gca 1472 Glu Leu Gln Lys Met Phe Thr Tyr Leu Met Glu Ser Glu Cys Lys Ala 345 350 355 tat aat cct aga cct ttc tgt aaa aca tac acc atg gat aag cag cct 1520 Tyr Asn Pro Arg Pro Phe Cys Lys Thr Tyr Thr Met Asp Lys Gln Pro 360 365 370 ctg aat act ggg gaa cag aaa gat atg aca gag ttt ttt act gat cta 1568 Leu Asn Thr Gly Glu Gln Lys Asp Met Thr Glu Phe Phe Thr Asp Leu 375 380 385 att acc aaa atc gaa gaa atg tct ccc gaa ctg aaa aat acc gtc aaa 1616 Ile Thr Lys Ile Glu Glu Met Ser Pro Glu Leu Lys Asn Thr Val Lys 390 395 400 agt tta ttt gga ggt gta att aca aac aat gtt gta tcc ttg gat tgt 1664 Ser Leu Phe Gly Gly Val Ile Thr Asn Asn Val Val Ser Leu Asp Cys 405 410 415 420 gaa cat gtt agt caa act gct gaa gag ttt tat act gtg agg tgc caa 1712 Glu His Val Ser Gln Thr Ala Glu Glu Phe Tyr Thr Val Arg Cys Gln 425 430 435 gtg gct gat atg aag aac att tat gaa tct ctt gat gaa gtt act ata 1760 Val Ala Asp Met Lys Asn Ile Tyr Glu Ser Leu Asp Glu Val Thr Ile 440 445 450 aaa gac act ttg gaa ggt gat aac atg tat act tgt tct cat tgt ggg 1808 Lys Asp Thr Leu Glu Gly Asp Asn Met Tyr Thr Cys Ser His Cys Gly 455 460 465 aag aaa gta cga gct gaa aaa agg gca tgt ttt aag aaa ttg cct cgc 1856 Lys Lys Val Arg Ala Glu Lys Arg Ala Cys Phe Lys Lys Leu Pro Arg 470 475 480 att ttg agt ttc aat act atg aga tac aca ttt aat atg gtc acg atg 1904 Ile Leu Ser Phe Asn Thr Met Arg Tyr Thr Phe Asn Met Val Thr Met 485 490 495 500 atg aaa gag aaa gtg aat aca cac ttt tcc ttc cca tta cgt ttg gac 1952 Met Lys Glu Lys Val Asn Thr His Phe Ser Phe Pro Leu Arg Leu Asp 505 510 515 atg acg ccc tat aca gaa gat ttt ctt atg gga aag agt gag agg aaa 2000 Met Thr Pro Tyr Thr Glu Asp Phe Leu Met Gly Lys Ser Glu Arg Lys 520 525 530 gaa ggt ttt aaa gaa gtc agt gat cat tca aaa gac tca gag agc tat 2048 Glu Gly Phe Lys Glu Val Ser Asp His Ser Lys Asp Ser Glu Ser Tyr 535 540 545 gaa tat gac ttg ata gga gtg act gtt cac aca gga acg gca gat ggt 2096 Glu Tyr Asp Leu Ile Gly Val Thr Val His Thr Gly Thr Ala Asp Gly 550 555 560 gga cac tat tat agc ttt atc aga gat ata gta aat ccc cat gct tat 2144 Gly His Tyr Tyr Ser Phe Ile Arg Asp Ile Val Asn Pro His Ala Tyr 565 570 575 580 aaa aac aat aaa tgg tat ctt ttt aat gat gct gag gta aaa cct ttt 2192 Lys Asn Asn Lys Trp Tyr Leu Phe Asn Asp Ala Glu Val Lys Pro Phe 585 590 595 gat tct gct caa ctt gca tct gaa tgt ttt ggt gga gag atg acg acc 2240 Asp Ser Ala Gln Leu Ala Ser Glu Cys Phe Gly Gly Glu Met Thr Thr 600 605 610 aag acc tat gat tct gtt aca gat aaa ttt atg gac ttc tct ttt gaa 2288 Lys Thr Tyr Asp Ser Val Thr Asp Lys Phe Met Asp Phe Ser Phe Glu 615 620 625 aag aca cac agt gca tat atg ctg ttt tac aaa cgc atg gaa cca gag 2336 Lys Thr His Ser Ala Tyr Met Leu Phe Tyr Lys Arg Met Glu Pro Glu 630 635 640 gaa gaa aat ggc aga gaa tac aaa ttt gat gtt tcg tca gag tta cta 2384 Glu Glu Asn Gly Arg Glu Tyr Lys Phe Asp Val Ser Ser Glu Leu Leu 645 650 655 660 gag tgg att tgg cat gat aac atg cag ttt ctt caa gac aaa aac att 2432 Glu Trp Ile Trp His Asp Asn Met Gln Phe Leu Gln Asp Lys Asn Ile 665 670 675 ttt gaa cat aca tat ttt gga ttt atg tgg caa ttg tgt agt tgt att 2480 Phe Glu His Thr Tyr Phe Gly Phe Met Trp Gln Leu Cys Ser Cys Ile 680 685 690 ccc agt aca tta cca gat cct aaa gct gtg tcc tta atg aca gca aag 2528 Pro Ser Thr Leu Pro Asp Pro Lys Ala Val Ser Leu Met Thr Ala Lys 695 700 705 tta agc act tcc ttt gtc cta gag aca ttt att cat tct aaa gaa aag 2576 Leu Ser Thr Ser Phe Val Leu Glu Thr Phe Ile His Ser Lys Glu Lys 710 715 720 ccc acg atg ctt cag tgg att gaa ctg ttg acg aaa cag ttt aat aat 2624 Pro Thr Met Leu Gln Trp Ile Glu Leu Leu Thr Lys Gln Phe Asn Asn 725 730 735 740 agt cag gca gct tgt gag tgg ttt tta gat cgt atg gct gat gac gac 2672 Ser Gln Ala Ala Cys Glu Trp Phe Leu Asp Arg Met Ala Asp Asp Asp 745 750 755 tgg tgg cca atg cag ata cta att aag tgc cct aat caa att gtg aga 2720 Trp Trp Pro Met Gln Ile Leu Ile Lys Cys Pro Asn Gln Ile Val Arg 760 765 770 cag atg ttt cag cgt ttg tgt atc cat gtg att cag agg ctg aga cct 2768 Gln Met Phe Gln Arg Leu Cys Ile His Val Ile Gln Arg Leu Arg Pro 775 780 785 gtg cat gct cat ctc tat ttg cag cca gga atg gaa gat ggg tca gat 2816 Val His Ala His Leu Tyr Leu Gln Pro Gly Met Glu Asp Gly Ser Asp 790 795 800 gat atg gat acc tca gta gaa gat att ggt ggt cgt tca tgt gtc act 2864 Asp Met Asp Thr Ser Val Glu Asp Ile Gly Gly Arg Ser Cys Val Thr 805 810 815 820 cgc ttt gtg aga acc ctg tta tta att atg gaa cat ggt gta aaa cct 2912 Arg Phe Val Arg Thr Leu Leu Leu Ile Met Glu His Gly Val Lys Pro 825 830 835 cac agt aaa cat ctt aca gag tat ttt gcc ttc ctt tac gaa ttt gca 2960 His Ser Lys His Leu Thr Glu Tyr Phe Ala Phe Leu Tyr Glu Phe Ala 840 845 850 aaa atg ggt gaa gaa gag agc caa ttt ttg ctt tca ttg caa gct ata 3008 Lys Met Gly Glu Glu Glu Ser Gln Phe Leu Leu Ser Leu Gln Ala Ile 855 860 865 tct aca atg gta cat ttt tac atg gga aca aaa gga cct gaa aat cct 3056 Ser Thr Met Val His Phe Tyr Met Gly Thr Lys Gly Pro Glu Asn Pro 870 875 880 caa gtt gaa gtg tta tca gag gaa gaa ggg gaa gaa gaa gag gag gaa 3104 Gln Val Glu Val Leu Ser Glu Glu Glu Gly Glu Glu Glu Glu Glu Glu 885 890 895 900 gaa gat atc ctc tct ctg gca gaa gaa aaa tac agg cca gct gcc ctt 3152 Glu Asp Ile Leu Ser Leu Ala Glu Glu Lys Tyr Arg Pro Ala Ala Leu 905 910 915 gaa aag atg ata gct tta gtt gct ctt ttg gtt gaa cag tct cga tca 3200 Glu Lys Met Ile Ala Leu Val Ala Leu Leu Val Glu Gln Ser Arg Ser 920 925 930 gaa agg tgaaatgttt cgaatttaaa atgtttaaag catgtttggt tttattattt 3256 Glu Arg ttacataatt gtttaccact agtttttcca ctagcttttt attatatatg tttaattatg 3316 taattgttat tcactagctt ttattatata aatcctttta aataatacta ctattcatca 3376 actcttgtgg cataagaatt tcagtttttt ctaccaaact tttacttcat ctatgagtcg 3436 tgttagaaat agtcattgaa aaaatataca gtaaaatatc taaaaaaaaa aaaaaagg 3494 2 934 PRT Homo sapiens 2 Met Ser Val Ala Tyr Thr Tyr Asp Asn Leu Ala Pro Arg Val Leu Lys 1 5 10 15 Ala Gln Ser Asp His Arg Ser Arg His Glu Val Ser His Tyr Ser Met 20 25 30 Trp Leu Leu Val Ser Trp Ala His Cys Cys Ser Leu Val Lys Ser Ser 35 40 45 Leu Ala Asp Ser Asp His Leu Gln Asp Trp Leu Lys Lys Leu Thr Leu 50 55 60 Leu Ile Pro Glu Thr Ala Val Arg His Glu Ser Cys Ser Gly Leu Tyr 65 70 75 80 Lys Leu Ser Leu Ser Gly Leu Asp Gly Gly Asp Ser Ile Asn Arg Ser 85 90 95 Phe Leu Leu Leu Ala Ala Ser Thr Leu Leu Lys Phe Leu Pro Asp Ala 100 105 110 Gln Ala Leu Lys Pro Ile Arg Ile Asp Asp Tyr Glu Glu Glu Pro Ile 115 120 125 Leu Lys Pro Gly Cys Lys Glu Tyr Phe Trp Leu Leu Cys Lys Leu Val 130 135 140 Asp Asn Ile His Ile Lys Asp Ala Ser Gln Thr Thr Leu Leu Asp Leu 145 150 155 160 Asp Ala Leu Ala Arg His Leu Ala Asp Cys Ile Arg Ser Arg Glu Ile 165 170 175 Leu Asp His Gln Asp Gly Asn Val Glu Asp Asp Gly Leu Thr Gly Leu 180 185 190 Leu Arg Leu Ala Thr Ser Val Val Lys His Lys Pro Pro Phe Lys Phe 195 200 205 Ser Arg Glu Gly Gln Glu Phe Leu Arg Asp Ile Phe Asn Leu Leu Phe 210 215 220 Leu Leu Pro Ser Leu Lys Asp Arg Gln Gln Pro Lys Cys Lys Ser His 225 230 235 240 Ser Thr Arg Ala Ala Ala Tyr Asp Leu Leu Val Glu Met Val Lys Gly 245 250 255 Ser Val Glu Asn Tyr Arg Leu Ile His Asn Trp Val Met Ala Gln His 260 265 270 Met Gln Ser His Ala Pro Tyr Lys Trp Asp Tyr Trp Pro His Glu Asp 275 280 285 Val Arg Ala Glu Cys Arg Phe Val Gly Leu Thr Asn Leu Gly Ala Thr 290 295 300 Cys Tyr Leu Ala Ser Thr Ile Gln Gln Leu Tyr Met Ile Pro Glu Ala 305 310 315 320 Arg Gln Ala Val Phe Thr Ala Lys Tyr Ser Glu Asp Met Lys His Lys 325 330 335 Thr Thr Leu Leu Glu Leu Gln Lys Met Phe Thr Tyr Leu Met Glu Ser 340 345 350 Glu Cys Lys Ala Tyr Asn Pro Arg Pro Phe Cys Lys Thr Tyr Thr Met 355 360 365 Asp Lys Gln Pro Leu Asn Thr Gly Glu Gln Lys Asp Met Thr Glu Phe 370 375 380 Phe Thr Asp Leu Ile Thr Lys Ile Glu Glu Met Ser Pro Glu Leu Lys 385 390 395 400 Asn Thr Val Lys Ser Leu Phe Gly Gly Val Ile Thr Asn Asn Val Val 405 410 415 Ser Leu Asp Cys Glu His Val Ser Gln Thr Ala Glu Glu Phe Tyr Thr 420 425 430 Val Arg Cys Gln Val Ala Asp Met Lys Asn Ile Tyr Glu Ser Leu Asp 435 440 445 Glu Val Thr Ile Lys Asp Thr Leu Glu Gly Asp Asn Met Tyr Thr Cys 450 455 460 Ser His Cys Gly Lys Lys Val Arg Ala Glu Lys Arg Ala Cys Phe Lys 465 470 475 480 Lys Leu Pro Arg Ile Leu Ser Phe Asn Thr Met Arg Tyr Thr Phe Asn 485 490 495 Met Val Thr Met Met Lys Glu Lys Val Asn Thr His Phe Ser Phe Pro 500 505 510 Leu Arg Leu Asp Met Thr Pro Tyr Thr Glu Asp Phe Leu Met Gly Lys 515 520 525 Ser Glu Arg Lys Glu Gly Phe Lys Glu Val Ser Asp His Ser Lys Asp 530 535 540 Ser Glu Ser Tyr Glu Tyr Asp Leu Ile Gly Val Thr Val His Thr Gly 545 550 555 560 Thr Ala Asp Gly Gly His Tyr Tyr Ser Phe Ile Arg Asp Ile Val Asn 565 570 575 Pro His Ala Tyr Lys Asn Asn Lys Trp Tyr Leu Phe Asn Asp Ala Glu 580 585 590 Val Lys Pro Phe Asp Ser Ala Gln Leu Ala Ser Glu Cys Phe Gly Gly 595 600 605 Glu Met Thr Thr Lys Thr Tyr Asp Ser Val Thr Asp Lys Phe Met Asp 610 615 620 Phe Ser Phe Glu Lys Thr His Ser Ala Tyr Met Leu Phe Tyr Lys Arg 625 630 635 640 Met Glu Pro Glu Glu Glu Asn Gly Arg Glu Tyr Lys Phe Asp Val Ser 645 650 655 Ser Glu Leu Leu Glu Trp Ile Trp His Asp Asn Met Gln Phe Leu Gln 660 665 670 Asp Lys Asn Ile Phe Glu His Thr Tyr Phe Gly Phe Met Trp Gln Leu 675 680 685 Cys Ser Cys Ile Pro Ser Thr Leu Pro Asp Pro Lys Ala Val Ser Leu 690 695 700 Met Thr Ala Lys Leu Ser Thr Ser Phe Val Leu Glu Thr Phe Ile His 705 710 715 720 Ser Lys Glu Lys Pro Thr Met Leu Gln Trp Ile Glu Leu Leu Thr Lys 725 730 735 Gln Phe Asn Asn Ser Gln Ala Ala Cys Glu Trp Phe Leu Asp Arg Met 740 745 750 Ala Asp Asp Asp Trp Trp Pro Met Gln Ile Leu Ile Lys Cys Pro Asn 755 760 765 Gln Ile Val Arg Gln Met Phe Gln Arg Leu Cys Ile His Val Ile Gln 770 775 780 Arg Leu Arg Pro Val His Ala His Leu Tyr Leu Gln Pro Gly Met Glu 785 790 795 800 Asp Gly Ser Asp Asp Met Asp Thr Ser Val Glu Asp Ile Gly Gly Arg 805 810 815 Ser Cys Val Thr Arg Phe Val Arg Thr Leu Leu Leu Ile Met Glu His 820 825 830 Gly Val Lys Pro His Ser Lys His Leu Thr Glu Tyr Phe Ala Phe Leu 835 840 845 Tyr Glu Phe Ala Lys Met Gly Glu Glu Glu Ser Gln Phe Leu Leu Ser 850 855 860 Leu Gln Ala Ile Ser Thr Met Val His Phe Tyr Met Gly Thr Lys Gly 865 870 875 880 Pro Glu Asn Pro Gln Val Glu Val Leu Ser Glu Glu Glu Gly Glu Glu 885 890 895 Glu Glu Glu Glu Glu Asp Ile Leu Ser Leu Ala Glu Glu Lys Tyr Arg 900 905 910 Pro Ala Ala Leu Glu Lys Met Ile Ala Leu Val Ala Leu Leu Val Glu 915 920 925 Gln Ser Arg Ser Glu Arg 930 3 2805 DNA Homo sapiens 3 atgtctgttg cttatacgta tgataatctg gctcctagag ttttaaaagc tcagtctgat 60 cacaggtcta gacatgaagt ttcacattat tcaatgtggc tcttggtgag ttgggctcat 120 tgctgttctt tagtgaaatc tagccttgct gatagcgatc atttacaaga ttggctaaag 180 aaattgactc tccttattcc tgagactgca gttcgtcatg aatcatgcag tggtctctat 240 aagttatccc tgtcagggct ggatggagga gactcaatca atcgttcttt tctgctattg 300 gctgcctcaa cattattgaa atttcttcct gatgctcaag cactcaaacc tattaggata 360 gatgattatg aggaagaacc aatattaaaa ccaggatgta aagagtattt ttggttgtta 420 tgcaaattag ttgacaacat acatataaag gacgctagtc agacaacgct cctcgactta 480 gatgccttgg caagacattt ggctgactgt attcgaagta gggagatcct tgatcatcag 540 gatggtaatg tagaagatga tgggcttaca ggactcctaa ggcttgcaac aagtgttgtt 600 aaacacaaac caccctttaa attttcaagg gaaggacagg aatttttgag agatatcttc 660 aatctcctgt ttttgttgcc aagtctaaag gaccgacaac agccaaagtg caaatcacat 720 tctacaagag ctgccgctta cgatttgtta gtagagatgg taaaggggtc tgttgagaac 780 tacaggctaa tacacaactg ggttatggca caacacatgc agtcccatgc accttataaa 840 tgggattact ggcctcatga agatgtccgt gctgaatgta gatttgttgg ccttactaac 900 cttggagcta cttgttactt agcttctact attcagcaac tttatatgat acctgaggca 960 agacaggctg tcttcactgc caagtattca gaggatatga agcacaagac cactcttctg 1020 gagcttcaga aaatgtttac atatttaatg gagagtgaat gcaaagcata taatcctaga 1080 cctttctgta aaacatacac catggataag cagcctctga atactgggga acagaaagat 1140 atgacagagt tttttactga tctaattacc aaaatcgaag aaatgtctcc cgaactgaaa 1200 aataccgtca aaagtttatt tggaggtgta attacaaaca atgttgtatc cttggattgt 1260 gaacatgtta gtcaaactgc tgaagagttt tatactgtga ggtgccaagt ggctgatatg 1320 aagaacattt atgaatctct tgatgaagtt actataaaag acactttgga aggtgataac 1380 atgtatactt gttctcattg tgggaagaaa gtacgagctg aaaaaagggc atgttttaag 1440 aaattgcctc gcattttgag tttcaatact atgagataca catttaatat ggtcacgatg 1500 atgaaagaga aagtgaatac acacttttcc ttcccattac gtttggacat gacgccctat 1560 acagaagatt ttcttatggg aaagagtgag aggaaagaag gttttaaaga agtcagtgat 1620 cattcaaaag actcagagag ctatgaatat gacttgatag gagtgactgt tcacacagga 1680 acggcagatg gtggacacta ttatagcttt atcagagata tagtaaatcc ccatgcttat 1740 aaaaacaata aatggtatct ttttaatgat gctgaggtaa aaccttttga ttctgctcaa 1800 cttgcatctg aatgttttgg tggagagatg acgaccaaga cctatgattc tgttacagat 1860 aaatttatgg acttctcttt tgaaaagaca cacagtgcat atatgctgtt ttacaaacgc 1920 atggaaccag aggaagaaaa tggcagagaa tacaaatttg atgtttcgtc agagttacta 1980 gagtggattt ggcatgataa catgcagttt cttcaagaca aaaacatttt tgaacataca 2040 tattttggat ttatgtggca attgtgtagt tgtattccca gtacattacc agatcctaaa 2100 gctgtgtcct taatgacagc aaagttaagc acttcctttg tcctagagac atttattcat 2160 tctaaagaaa agcccacgat gcttcagtgg attgaactgt tgacgaaaca gtttaataat 2220 agtcaggcag cttgtgagtg gtttttagat cgtatggctg atgacgactg gtggccaatg 2280 cagatactaa ttaagtgccc taatcaaatt gtgagacaga tgtttcagcg tttgtgtatc 2340 catgtgattc agaggctgag acctgtgcat gctcatctct atttgcagcc aggaatggaa 2400 gatgggtcag atgatatgga tacctcagta gaagatattg gtggtcgttc atgtgtcact 2460 cgctttgtga gaaccctgtt attaattatg gaacatggtg taaaacctca cagtaaacat 2520 cttacagagt attttgcctt cctttacgaa tttgcaaaaa tgggtgaaga agagagccaa 2580 tttttgcttt cattgcaagc tatatctaca atggtacatt tttacatggg aacaaaagga 2640 cctgaaaatc ctcaagttga agtgttatca gaggaagaag gggaagaaga agaggaggaa 2700 gaagatatcc tctctctggc agaagaaaaa tacaggccag ctgcccttga aaagatgata 2760 gctttagttg ctcttttggt tgaacagtct cgatcagaaa ggtga 2805 4 4873 DNA Homo sapiens CDS (85)...(3501) 4 ccacgcgtcc ggcctagtcc tgagaggctg ggccggcggc ggctgcggcg ggagaccggt 60 gacccgcggc tgggcgcctc ggcc atg act gcg gag ctg cag cag gac gac 111 Met Thr Ala Glu Leu Gln Gln Asp Asp 1 5 gcg gcc ggc gcg gca gac ggc cac ggc tcg agc tgc caa atg ctg tta 159 Ala Ala Gly Ala Ala Asp Gly His Gly Ser Ser Cys Gln Met Leu Leu 10 15 20 25 aat caa ctg aga gaa atc aca ggc att cag gac cct tcc ttt ctc cat 207 Asn Gln Leu Arg Glu Ile Thr Gly Ile Gln Asp Pro Ser Phe Leu His 30 35 40 gaa gct ctg aag gcc agt aat ggt gac att act cag gca gtc agc ctt 255 Glu Ala Leu Lys Ala Ser Asn Gly Asp Ile Thr Gln Ala Val Ser Leu 45 50 55 ctc act gat gag aga gtt aag gag ccc agt caa gac act gtt gct aca 303 Leu Thr Asp Glu Arg Val Lys Glu Pro Ser Gln Asp Thr Val Ala Thr 60 65 70 gaa cca tct gaa gta gag ggg agt gct gcc aac aag gaa gta tta gca 351 Glu Pro Ser Glu Val Glu Gly Ser Ala Ala Asn Lys Glu Val Leu Ala 75 80 85 aaa gtt ata gac ctt act cat gat aac aaa gat gat ctt cag gct gcc 399 Lys Val Ile Asp Leu Thr His Asp Asn Lys Asp Asp Leu Gln Ala Ala 90 95 100 105 att gct ttg agt cta ctg gag tct ccc aaa att caa gct gat gga aga 447 Ile Ala Leu Ser Leu Leu Glu Ser Pro Lys Ile Gln Ala Asp Gly Arg 110 115 120 gat ctt aac agg atg cat gaa gca acc tct gca gaa act aaa cgc tca 495 Asp Leu Asn Arg Met His Glu Ala Thr Ser Ala Glu Thr Lys Arg Ser 125 130 135 aag aga aaa cgc tgt gaa gtc tgg gga gaa aac ccc aat ccc aat gac 543 Lys Arg Lys Arg Cys Glu Val Trp Gly Glu Asn Pro Asn Pro Asn Asp 140 145 150 tgg agg aga gtt gat ggt tgg cca gtt ggg ctg aaa aat gtt ggc aat 591 Trp Arg Arg Val Asp Gly Trp Pro Val Gly Leu Lys Asn Val Gly Asn 155 160 165 aca tgt tgg ttt agt gct gtt att cag tct ctc ttt caa ttg cct gaa 639 Thr Cys Trp Phe Ser Ala Val Ile Gln Ser Leu Phe Gln Leu Pro Glu 170 175 180 185 ttt cga aga ctt gtt ctc agt tat agt ctg cca caa aat gta ctt gaa 687 Phe Arg Arg Leu Val Leu Ser Tyr Ser Leu Pro Gln Asn Val Leu Glu 190 195 200 aat tgt cga agt cat aca gaa aag aga aat atc atg ttt atg caa gag 735 Asn Cys Arg Ser His Thr Glu Lys Arg Asn Ile Met Phe Met Gln Glu 205 210 215 ctt cag tat ttg ttt gct cta atg atg gga tca aat aga aaa ttt gta 783 Leu Gln Tyr Leu Phe Ala Leu Met Met Gly Ser Asn Arg Lys Phe Val 220 225 230 gac ccg tct gca gcc ctg gat cta tta aag gga gca ttc cga tca tct 831 Asp Pro Ser Ala Ala Leu Asp Leu Leu Lys Gly Ala Phe Arg Ser Ser 235 240 245 gag gaa cag cag caa gat gtg agt gaa ttc aca cac aag ctc ctg gat 879 Glu Glu Gln Gln Gln Asp Val Ser Glu Phe Thr His Lys Leu Leu Asp 250 255 260 265 tgg cta gag gac gca ttc cag cta gct gtt aat gtt aac agt ccc agg 927 Trp Leu Glu Asp Ala Phe Gln Leu Ala Val Asn Val Asn Ser Pro Arg 270 275 280 aac aaa tct gaa aat cca atg gtg cag ctg ttc tat ggt act ttc ctg 975 Asn Lys Ser Glu Asn Pro Met Val Gln Leu Phe Tyr Gly Thr Phe Leu 285 290 295 act gaa ggg gtt cgt gaa gga aaa ccc ttt tgt aac aat gag acc ttc 1023 Thr Glu Gly Val Arg Glu Gly Lys Pro Phe Cys Asn Asn Glu Thr Phe 300 305 310 ggc cag tat cct ctt cag gta aac ggt tat cgc aac tta gac gag tgt 1071 Gly Gln Tyr Pro Leu Gln Val Asn Gly Tyr Arg Asn Leu Asp Glu Cys 315 320 325 ttg gaa ggg gcc atg gtg gag ggt gat gtt gag ctt ctt ccc tcc gat 1119 Leu Glu Gly Ala Met Val Glu Gly Asp Val Glu Leu Leu Pro Ser Asp 330 335 340 345 cac tcg gtg aag tat gga caa gag cgt tgg ttt aca aag cta cct cca 1167 His Ser Val Lys Tyr Gly Gln Glu Arg Trp Phe Thr Lys Leu Pro Pro 350 355 360 gtg ttg acc ttt gaa ctc tca aga ttt gag ttt aat cag tcc ctt ggg 1215 Val Leu Thr Phe Glu Leu Ser Arg Phe Glu Phe Asn Gln Ser Leu Gly 365 370 375 cag cca gag aaa att cac aat aag ctg gaa ttt cct cag att att tat 1263 Gln Pro Glu Lys Ile His Asn Lys Leu Glu Phe Pro Gln Ile Ile Tyr 380 385 390 atg gac agg tac atg tac agg agc aag gag ctt att cga aat aag aga 1311 Met Asp Arg Tyr Met Tyr Arg Ser Lys Glu Leu Ile Arg Asn Lys Arg 395 400 405 gag tgt att cga aag ttg aag gag gaa ata aaa att ctg cag caa aaa 1359 Glu Cys Ile Arg Lys Leu Lys Glu Glu Ile Lys Ile Leu Gln Gln Lys 410 415 420 425 ttg gaa agg tat gtg aaa tat ggc tca ggc cca gct cgg ttc ccg ctc 1407 Leu Glu Arg Tyr Val Lys Tyr Gly Ser Gly Pro Ala Arg Phe Pro Leu 430 435 440 ccg gac atg ctg aaa tat gtt att gaa ttt gct agt aca aaa cct gcc 1455 Pro Asp Met Leu Lys Tyr Val Ile Glu Phe Ala Ser Thr Lys Pro Ala 445 450 455 tca gaa agc tgt cca cct gaa agt gac aca cat atg aca tta cca ctt 1503 Ser Glu Ser Cys Pro Pro Glu Ser Asp Thr His Met Thr Leu Pro Leu 460 465 470 tct tca gtg cac tgc tcg gtt tct gac cag aca tcc aag gaa agt aca 1551 Ser Ser Val His Cys Ser Val Ser Asp Gln Thr Ser Lys Glu Ser Thr 475 480 485 agt aca gaa agc tct tct cag gat gtt gaa agt acc ttt tct tct cct 1599 Ser Thr Glu Ser Ser Ser Gln Asp Val Glu Ser Thr Phe Ser Ser Pro 490 495 500 505 gaa gat tct tta ccc aag tct aaa cca ctg aca tct tct cgg tct tcc 1647 Glu Asp Ser Leu Pro Lys Ser Lys Pro Leu Thr Ser Ser Arg Ser Ser 510 515 520 atg gaa atg cct tca cag cca gct cca cga aca gtc aca gat gag gag 1695 Met Glu Met Pro Ser Gln Pro Ala Pro Arg Thr Val Thr Asp Glu Glu 525 530 535 ata aat ttt gtt aag acc tgt ctt cag aga tgg agg agt gag att gaa 1743 Ile Asn Phe Val Lys Thr Cys Leu Gln Arg Trp Arg Ser Glu Ile Glu 540 545 550 caa gat ata caa gat tta aag act tgt att gca agt act act cag act 1791 Gln Asp Ile Gln Asp Leu Lys Thr Cys Ile Ala Ser Thr Thr Gln Thr 555 560 565 att gaa cag atg tac tgc gat cct ctc ctt cgt cag gtg cct tat cgc 1839 Ile Glu Gln Met Tyr Cys Asp Pro Leu Leu Arg Gln Val Pro Tyr Arg 570 575 580 585 ttg cat gca gtt ctt gtt cat gaa gga caa gca aat gct gga cac tat 1887 Leu His Ala Val Leu Val His Glu Gly Gln Ala Asn Ala Gly His Tyr 590 595 600 tgg gcc tat atc tat aat caa ccc cga cag agc tgg ctc aag tac aat 1935 Trp Ala Tyr Ile Tyr Asn Gln Pro Arg Gln Ser Trp Leu Lys Tyr Asn 605 610 615 gac atc tct gtt act gaa tct tcc tgg gaa gaa gtt gaa aga gat tcc 1983 Asp Ile Ser Val Thr Glu Ser Ser Trp Glu Glu Val Glu Arg Asp Ser 620 625 630 tat gga ggc ctg aga aat gtt agt gct tac tgt ctg atg tac att aat 2031 Tyr Gly Gly Leu Arg Asn Val Ser Ala Tyr Cys Leu Met Tyr Ile Asn 635 640 645 gac aaa cta ccc tac ttc aat gca gag gca gcc cca act gaa tca gat 2079 Asp Lys Leu Pro Tyr Phe Asn Ala Glu Ala Ala Pro Thr Glu Ser Asp 650 655 660 665 caa atg tca gaa gtg gaa gcc cta tct gtg gaa ctc aag cat tac att 2127 Gln Met Ser Glu Val Glu Ala Leu Ser Val Glu Leu Lys His Tyr Ile 670 675 680 cag gag gat aac tgg cgg ttt gag cag gaa gta gag gag tgg gaa gaa 2175 Gln Glu Asp Asn Trp Arg Phe Glu Gln Glu Val Glu Glu Trp Glu Glu 685 690 695 gag cag tct tgc aaa atc cct caa atg gag tcc tcc acc aac tcc tca 2223 Glu Gln Ser Cys Lys Ile Pro Gln Met Glu Ser Ser Thr Asn Ser Ser 700 705 710 tca cag gac tac tct aca tca caa gag cct tca gta gcc tct tct cat 2271 Ser Gln Asp Tyr Ser Thr Ser Gln Glu Pro Ser Val Ala Ser Ser His 715 720 725 ggg gtt cgc tgc ttg tcg tct gag cat gct gtg att gta aag gag caa 2319 Gly Val Arg Cys Leu Ser Ser Glu His Ala Val Ile Val Lys Glu Gln 730 735 740 745 act gcc cag gct att gca aac aca gcc cgt gcc tat gag aag agc ggt 2367 Thr Ala Gln Ala Ile Ala Asn Thr Ala Arg Ala Tyr Glu Lys Ser Gly 750 755 760 gta gaa gcg gca ctg agt gag gtt aaa gaa gct gaa ccc aag aag ccc 2415 Val Glu Ala Ala Leu Ser Glu Val Lys Glu Ala Glu Pro Lys Lys Pro 765 770 775 atg ccc cag gaa aca aac ctt gca gag cag tca gaa cag ccc cca aag 2463 Met Pro Gln Glu Thr Asn Leu Ala Glu Gln Ser Glu Gln Pro Pro Lys 780 785 790 gct aat gat gca gag tct act gcc cag cct aat tct gag gtc tct gaa 2511 Ala Asn Asp Ala Glu Ser Thr Ala Gln Pro Asn Ser Glu Val Ser Glu 795 800 805 gtc gag att ccc agt gtg gga agg att ctg gtt aga tct gat gca gat 2559 Val Glu Ile Pro Ser Val Gly Arg Ile Leu Val Arg Ser Asp Ala Asp 810 815 820 825 gga tat gat gag gag gtg atg ctg agc cct gcc atg caa ggg gtc atc 2607 Gly Tyr Asp Glu Glu Val Met Leu Ser Pro Ala Met Gln Gly Val Ile 830 835 840 ctg gcc ata gct aaa gcc cgt cag acc ttt gac cga gat ggg tct gaa 2655 Leu Ala Ile Ala Lys Ala Arg Gln Thr Phe Asp Arg Asp Gly Ser Glu 845 850 855 gca ggg ctg att aag gca ttc cat gaa gaa tac tcc agg ctc tat cag 2703 Ala Gly Leu Ile Lys Ala Phe His Glu Glu Tyr Ser Arg Leu Tyr Gln 860 865 870 ctt gcc aaa gag acc ccc acc tct cac agt gat cct cga ctt cag cat 2751 Leu Ala Lys Glu Thr Pro Thr Ser His Ser Asp Pro Arg Leu Gln His 875 880 885 gtc ctt gtc tac ttt ttc caa aat gaa gca ccc aaa agg gta gta gaa 2799 Val Leu Val Tyr Phe Phe Gln Asn Glu Ala Pro Lys Arg Val Val Glu 890 895 900 905 cga acc ctt ctg gaa cag ttt gca gat aaa aat ctt agc tat gat gaa 2847 Arg Thr Leu Leu Glu Gln Phe Ala Asp Lys Asn Leu Ser Tyr Asp Glu 910 915 920 aga tca atc agc att atg aag gtg gct caa gcg aaa ctg aag gaa att 2895 Arg Ser Ile Ser Ile Met Lys Val Ala Gln Ala Lys Leu Lys Glu Ile 925 930 935 ggt cca gat gac atg aat atg gaa gag tac aag aag tgg cat gaa gat 2943 Gly Pro Asp Asp Met Asn Met Glu Glu Tyr Lys Lys Trp His Glu Asp 940 945 950 tat agt ttg ttc cga aaa gtg tct gtg tat ctc cta aca ggc cta gaa 2991 Tyr Ser Leu Phe Arg Lys Val Ser Val Tyr Leu Leu Thr Gly Leu Glu 955 960 965 ctc tat caa aaa gga aag tac caa gag gca ctt tcc tac ctg gta tat 3039 Leu Tyr Gln Lys Gly Lys Tyr Gln Glu Ala Leu Ser Tyr Leu Val Tyr 970 975 980 985 gcc tac cag agc aat gct gcc ctg ctg atg aag ggg ccc cgc cgg ggg 3087 Ala Tyr Gln Ser Asn Ala Ala Leu Leu Met Lys Gly Pro Arg Arg Gly 990 995 1000 gtc aaa gaa tcc gtg att gct tta tac cga aga aaa tgc ctt ctg gag 3135 Val Lys Glu Ser Val Ile Ala Leu Tyr Arg Arg Lys Cys Leu Leu Glu 1005 1010 1015 ctg aat gcc aaa gca gct tct ctt ttt gaa aca aat gat gat cac tcc 3183 Leu Asn Ala Lys Ala Ala Ser Leu Phe Glu Thr Asn Asp Asp His Ser 1020 1025 1030 gta act gag ggc att aat gtg atg aat gaa ctg atc atc ccc tgc att 3231 Val Thr Glu Gly Ile Asn Val Met Asn Glu Leu Ile Ile Pro Cys Ile 1035 1040 1045 cac ctt atc att aat aat gac att tcc aag gat gat ctg gat gcc att 3279 His Leu Ile Ile Asn Asn Asp Ile Ser Lys Asp Asp Leu Asp Ala Ile 1050 1055 1060 1065 gag gtc atg aga aac cat tgg tgc tct tac ctt ggg caa gat att gca 3327 Glu Val Met Arg Asn His Trp Cys Ser Tyr Leu Gly Gln Asp Ile Ala 1070 1075 1080 gaa aat ctg cag ctg tgc cta ggg gag ttt cta ccc aga ctt cta gat 3375 Glu Asn Leu Gln Leu Cys Leu Gly Glu Phe Leu Pro Arg Leu Leu Asp 1085 1090 1095 cct tct gca gaa atc atc gtc ttg aaa gag cct cca act att cga ccc 3423 Pro Ser Ala Glu Ile Ile Val Leu Lys Glu Pro Pro Thr Ile Arg Pro 1100 1105 1110 aat tct ccc tat gac cta tgt agc cga ttt gca gct gtc atg gag tca 3471 Asn Ser Pro Tyr Asp Leu Cys Ser Arg Phe Ala Ala Val Met Glu Ser 1115 1120 1125 att cag gga gtt tca act gtg aca gtg aaa taagctccca catgttcaag 3521 Ile Gln Gly Val Ser Thr Val Thr Val Lys 1130 1135 gcccattctg gttcctggct gcctgcctct tgcacagaag ttcgttgtca tagtgctcac 3581 cttgggaaaa ggattaggtg ggcacataag attccgatca gaccccaacc atgctgcatg 3641 tgtaaagaag gattgaaaat aaaattgcac tttttaggta caaaatcata aaagctgttt 3701 cactagaaaa ggcagaaagc agtgtattaa ggtgttgaat tacgccagaa gacctgaaat 3761 gccttgtacc tacaacaatg cttaggcttt tctaagcctc ttgccacttt taaaattatc 3821 cttcaggcat aaatattttt gacagcagaa tagaagaatg attcatgaga acctgaacca 3881 gatgaacagc tactagttat tttatcaaat acagatgaca tttaaaaatt cttaactaca 3941 agagattaga aatataaacc ttgcctggct cttgccagga gataacaaaa tgggttgctg 4001 atgaactgca cccttttaca tgtgggtaga atataagctc acatggcagt gagatgttga 4061 aaagtcaaaa gagacctgtc tctctccttt cttttctatc tttaaaccag aaaacctcat 4121 actcagtcct cagtgaaaga aagtaaagta ttaaggactt taggcagaag agcattgtgt 4181 aacttgactg aagatcatcc attaatagtt attaggcatt taggtaaaat tttctaatac 4241 ctaaaaattg tcaaaaacag tcaatagggc tactgctggc ccaaagacca tttaggtcca 4301 cctcctcttt tttgctcttt tttttttttc tgtgacagtt tcactgtgtc gcccaggctg 4361 gcgttcagtg gtgcaatctc agctcactgc aaactctgtc tcctgggctc aagtgattct 4421 cgtgcctcag cctcccgaat agctggaatt acgggcatgc accaccacac ctggctaatt 4481 tttgtatttt taatagagat ggggtttcac catattggcc aggctgatct ctaactcctg 4541 gcctcaagtg atctatctgc ctccctcagc ctcccaaagt ctgggattgc agacaagtca 4601 tcgtacccgg ccttcttttt tgcccttaaa agtaagggat gtgggtttgt acaaaaaaaa 4661 aaaaaaaaaa aaaaaaaaac cagcatacat atgcaaaact atatatatat gtatatgtag 4721 agaaaaatac ttcccattga tcatttttaa aaggcttctg attggatatt gtgttttaac 4781 caaattttaa agattaatgg aatcatgaaa gggaaaaaat tgatacaact atgcagattt 4841 tataaatgtg caataaaagt atttgtttta ca 4873 5 1139 PRT Homo sapiens 5 Met Thr Ala Glu Leu Gln Gln Asp Asp Ala Ala Gly Ala Ala Asp Gly 1 5 10 15 His Gly Ser Ser Cys Gln Met Leu Leu Asn Gln Leu Arg Glu Ile Thr 20 25 30 Gly Ile Gln Asp Pro Ser Phe Leu His Glu Ala Leu Lys Ala Ser Asn 35 40 45 Gly Asp Ile Thr Gln Ala Val Ser Leu Leu Thr Asp Glu Arg Val Lys 50 55 60 Glu Pro Ser Gln Asp Thr Val Ala Thr Glu Pro Ser Glu Val Glu Gly 65 70 75 80 Ser Ala Ala Asn Lys Glu Val Leu Ala Lys Val Ile Asp Leu Thr His 85 90 95 Asp Asn Lys Asp Asp Leu Gln Ala Ala Ile Ala Leu Ser Leu Leu Glu 100 105 110 Ser Pro Lys Ile Gln Ala Asp Gly Arg Asp Leu Asn Arg Met His Glu 115 120 125 Ala Thr Ser Ala Glu Thr Lys Arg Ser Lys Arg Lys Arg Cys Glu Val 130 135 140 Trp Gly Glu Asn Pro Asn Pro Asn Asp Trp Arg Arg Val Asp Gly Trp 145 150 155 160 Pro Val Gly Leu Lys Asn Val Gly Asn Thr Cys Trp Phe Ser Ala Val 165 170 175 Ile Gln Ser Leu Phe Gln Leu Pro Glu Phe Arg Arg Leu Val Leu Ser 180 185 190 Tyr Ser Leu Pro Gln Asn Val Leu Glu Asn Cys Arg Ser His Thr Glu 195 200 205 Lys Arg Asn Ile Met Phe Met Gln Glu Leu Gln Tyr Leu Phe Ala Leu 210 215 220 Met Met Gly Ser Asn Arg Lys Phe Val Asp Pro Ser Ala Ala Leu Asp 225 230 235 240 Leu Leu Lys Gly Ala Phe Arg Ser Ser Glu Glu Gln Gln Gln Asp Val 245 250 255 Ser Glu Phe Thr His Lys Leu Leu Asp Trp Leu Glu Asp Ala Phe Gln 260 265 270 Leu Ala Val Asn Val Asn Ser Pro Arg Asn Lys Ser Glu Asn Pro Met 275 280 285 Val Gln Leu Phe Tyr Gly Thr Phe Leu Thr Glu Gly Val Arg Glu Gly 290 295 300 Lys Pro Phe Cys Asn Asn Glu Thr Phe Gly Gln Tyr Pro Leu Gln Val 305 310 315 320 Asn Gly Tyr Arg Asn Leu Asp Glu Cys Leu Glu Gly Ala Met Val Glu 325 330 335 Gly Asp Val Glu Leu Leu Pro Ser Asp His Ser Val Lys Tyr Gly Gln 340 345 350 Glu Arg Trp Phe Thr Lys Leu Pro Pro Val Leu Thr Phe Glu Leu Ser 355 360 365 Arg Phe Glu Phe Asn Gln Ser Leu Gly Gln Pro Glu Lys Ile His Asn 370 375 380 Lys Leu Glu Phe Pro Gln Ile Ile Tyr Met Asp Arg Tyr Met Tyr Arg 385 390 395 400 Ser Lys Glu Leu Ile Arg Asn Lys Arg Glu Cys Ile Arg Lys Leu Lys 405 410 415 Glu Glu Ile Lys Ile Leu Gln Gln Lys Leu Glu Arg Tyr Val Lys Tyr 420 425 430 Gly Ser Gly Pro Ala Arg Phe Pro Leu Pro Asp Met Leu Lys Tyr Val 435 440 445 Ile Glu Phe Ala Ser Thr Lys Pro Ala Ser Glu Ser Cys Pro Pro Glu 450 455 460 Ser Asp Thr His Met Thr Leu Pro Leu Ser Ser Val His Cys Ser Val 465 470 475 480 Ser Asp Gln Thr Ser Lys Glu Ser Thr Ser Thr Glu Ser Ser Ser Gln 485 490 495 Asp Val Glu Ser Thr Phe Ser Ser Pro Glu Asp Ser Leu Pro Lys Ser 500 505 510 Lys Pro Leu Thr Ser Ser Arg Ser Ser Met Glu Met Pro Ser Gln Pro 515 520 525 Ala Pro Arg Thr Val Thr Asp Glu Glu Ile Asn Phe Val Lys Thr Cys 530 535 540 Leu Gln Arg Trp Arg Ser Glu Ile Glu Gln Asp Ile Gln Asp Leu Lys 545 550 555 560 Thr Cys Ile Ala Ser Thr Thr Gln Thr Ile Glu Gln Met Tyr Cys Asp 565 570 575 Pro Leu Leu Arg Gln Val Pro Tyr Arg Leu His Ala Val Leu Val His 580 585 590 Glu Gly Gln Ala Asn Ala Gly His Tyr Trp Ala Tyr Ile Tyr Asn Gln 595 600 605 Pro Arg Gln Ser Trp Leu Lys Tyr Asn Asp Ile Ser Val Thr Glu Ser 610 615 620 Ser Trp Glu Glu Val Glu Arg Asp Ser Tyr Gly Gly Leu Arg Asn Val 625 630 635 640 Ser Ala Tyr Cys Leu Met Tyr Ile Asn Asp Lys Leu Pro Tyr Phe Asn 645 650 655 Ala Glu Ala Ala Pro Thr Glu Ser Asp Gln Met Ser Glu Val Glu Ala 660 665 670 Leu Ser Val Glu Leu Lys His Tyr Ile Gln Glu Asp Asn Trp Arg Phe 675 680 685 Glu Gln Glu Val Glu Glu Trp Glu Glu Glu Gln Ser Cys Lys Ile Pro 690 695 700 Gln Met Glu Ser Ser Thr Asn Ser Ser Ser Gln Asp Tyr Ser Thr Ser 705 710 715 720 Gln Glu Pro Ser Val Ala Ser Ser His Gly Val Arg Cys Leu Ser Ser 725 730 735 Glu His Ala Val Ile Val Lys Glu Gln Thr Ala Gln Ala Ile Ala Asn 740 745 750 Thr Ala Arg Ala Tyr Glu Lys Ser Gly Val Glu Ala Ala Leu Ser Glu 755 760 765 Val Lys Glu Ala Glu Pro Lys Lys Pro Met Pro Gln Glu Thr Asn Leu 770 775 780 Ala Glu Gln Ser Glu Gln Pro Pro Lys Ala Asn Asp Ala Glu Ser Thr 785 790 795 800 Ala Gln Pro Asn Ser Glu Val Ser Glu Val Glu Ile Pro Ser Val Gly 805 810 815 Arg Ile Leu Val Arg Ser Asp Ala Asp Gly Tyr Asp Glu Glu Val Met 820 825 830 Leu Ser Pro Ala Met Gln Gly Val Ile Leu Ala Ile Ala Lys Ala Arg 835 840 845 Gln Thr Phe Asp Arg Asp Gly Ser Glu Ala Gly Leu Ile Lys Ala Phe 850 855 860 His Glu Glu Tyr Ser Arg Leu Tyr Gln Leu Ala Lys Glu Thr Pro Thr 865 870 875 880 Ser His Ser Asp Pro Arg Leu Gln His Val Leu Val Tyr Phe Phe Gln 885 890 895 Asn Glu Ala Pro Lys Arg Val Val Glu Arg Thr Leu Leu Glu Gln Phe 900 905 910 Ala Asp Lys Asn Leu Ser Tyr Asp Glu Arg Ser Ile Ser Ile Met Lys 915 920 925 Val Ala Gln Ala Lys Leu Lys Glu Ile Gly Pro Asp Asp Met Asn Met 930 935 940 Glu Glu Tyr Lys Lys Trp His Glu Asp Tyr Ser Leu Phe Arg Lys Val 945 950 955 960 Ser Val Tyr Leu Leu Thr Gly Leu Glu Leu Tyr Gln Lys Gly Lys Tyr 965 970 975 Gln Glu Ala Leu Ser Tyr Leu Val Tyr Ala Tyr Gln Ser Asn Ala Ala 980 985 990 Leu Leu Met Lys Gly Pro Arg Arg Gly Val Lys Glu Ser Val Ile Ala 995 1000 1005 Leu Tyr Arg Arg Lys Cys Leu Leu Glu Leu Asn Ala Lys Ala Ala Ser 1010 1015 1020 Leu Phe Glu Thr Asn Asp Asp His Ser Val Thr Glu Gly Ile Asn Val 1025 1030 1035 1040 Met Asn Glu Leu Ile Ile Pro Cys Ile His Leu Ile Ile Asn Asn Asp 1045 1050 1055 Ile Ser Lys Asp Asp Leu Asp Ala Ile Glu Val Met Arg Asn His Trp 1060 1065 1070 Cys Ser Tyr Leu Gly Gln Asp Ile Ala Glu Asn Leu Gln Leu Cys Leu 1075 1080 1085 Gly Glu Phe Leu Pro Arg Leu Leu Asp Pro Ser Ala Glu Ile Ile Val 1090 1095 1100 Leu Lys Glu Pro Pro Thr Ile Arg Pro Asn Ser Pro Tyr Asp Leu Cys 1105 1110 1115 1120 Ser Arg Phe Ala Ala Val Met Glu Ser Ile Gln Gly Val Ser Thr Val 1125 1130 1135 Thr Val Lys 6 3420 DNA Homo sapiens 6 atgactgcgg agctgcagca ggacgacgcg gccggcgcgg cagacggcca cggctcgagc 60 tgccaaatgc tgttaaatca actgagagaa atcacaggca ttcaggaccc ttcctttctc 120 catgaagctc tgaaggccag taatggtgac attactcagg cagtcagcct tctcactgat 180 gagagagtta aggagcccag tcaagacact gttgctacag aaccatctga agtagagggg 240 agtgctgcca acaaggaagt attagcaaaa gttatagacc ttactcatga taacaaagat 300 gatcttcagg ctgccattgc tttgagtcta ctggagtctc ccaaaattca agctgatgga 360 agagatctta acaggatgca tgaagcaacc tctgcagaaa ctaaacgctc aaagagaaaa 420 cgctgtgaag tctggggaga aaaccccaat cccaatgact ggaggagagt tgatggttgg 480 ccagttgggc tgaaaaatgt tggcaataca tgttggttta gtgctgttat tcagtctctc 540 tttcaattgc ctgaatttcg aagacttgtt ctcagttata gtctgccaca aaatgtactt 600 gaaaattgtc gaagtcatac agaaaagaga aatatcatgt ttatgcaaga gcttcagtat 660 ttgtttgctc taatgatggg atcaaataga aaatttgtag acccgtctgc agccctggat 720 ctattaaagg gagcattccg atcatctgag gaacagcagc aagatgtgag tgaattcaca 780 cacaagctcc tggattggct agaggacgca ttccagctag ctgttaatgt taacagtccc 840 aggaacaaat ctgaaaatcc aatggtgcag ctgttctatg gtactttcct gactgaaggg 900 gttcgtgaag gaaaaccctt ttgtaacaat gagaccttcg gccagtatcc tcttcaggta 960 aacggttatc gcaacttaga cgagtgtttg gaaggggcca tggtggaggg tgatgttgag 1020 cttcttccct ccgatcactc ggtgaagtat ggacaagagc gttggtttac aaagctacct 1080 ccagtgttga cctttgaact ctcaagattt gagtttaatc agtcccttgg gcagccagag 1140 aaaattcaca ataagctgga atttcctcag attatttata tggacaggta catgtacagg 1200 agcaaggagc ttattcgaaa taagagagag tgtattcgaa agttgaagga ggaaataaaa 1260 attctgcagc aaaaattgga aaggtatgtg aaatatggct caggcccagc tcggttcccg 1320 ctcccggaca tgctgaaata tgttattgaa tttgctagta caaaacctgc ctcagaaagc 1380 tgtccacctg aaagtgacac acatatgaca ttaccacttt cttcagtgca ctgctcggtt 1440 tctgaccaga catccaagga aagtacaagt acagaaagct cttctcagga tgttgaaagt 1500 accttttctt ctcctgaaga ttctttaccc aagtctaaac cactgacatc ttctcggtct 1560 tccatggaaa tgccttcaca gccagctcca cgaacagtca cagatgagga gataaatttt 1620 gttaagacct gtcttcagag atggaggagt gagattgaac aagatataca agatttaaag 1680 acttgtattg caagtactac tcagactatt gaacagatgt actgcgatcc tctccttcgt 1740 caggtgcctt atcgcttgca tgcagttctt gttcatgaag gacaagcaaa tgctggacac 1800 tattgggcct atatctataa tcaaccccga cagagctggc tcaagtacaa tgacatctct 1860 gttactgaat cttcctggga agaagttgaa agagattcct atggaggcct gagaaatgtt 1920 agtgcttact gtctgatgta cattaatgac aaactaccct acttcaatgc agaggcagcc 1980 ccaactgaat cagatcaaat gtcagaagtg gaagccctat ctgtggaact caagcattac 2040 attcaggagg ataactggcg gtttgagcag gaagtagagg agtgggaaga agagcagtct 2100 tgcaaaatcc ctcaaatgga gtcctccacc aactcctcat cacaggacta ctctacatca 2160 caagagcctt cagtagcctc ttctcatggg gttcgctgct tgtcgtctga gcatgctgtg 2220 attgtaaagg agcaaactgc ccaggctatt gcaaacacag cccgtgccta tgagaagagc 2280 ggtgtagaag cggcactgag tgaggttaaa gaagctgaac ccaagaagcc catgccccag 2340 gaaacaaacc ttgcagagca gtcagaacag cccccaaagg ctaatgatgc agagtctact 2400 gcccagccta attctgaggt ctctgaagtc gagattccca gtgtgggaag gattctggtt 2460 agatctgatg cagatggata tgatgaggag gtgatgctga gccctgccat gcaaggggtc 2520 atcctggcca tagctaaagc ccgtcagacc tttgaccgag atgggtctga agcagggctg 2580 attaaggcat tccatgaaga atactccagg ctctatcagc ttgccaaaga gacccccacc 2640 tctcacagtg atcctcgact tcagcatgtc cttgtctact ttttccaaaa tgaagcaccc 2700 aaaagggtag tagaacgaac ccttctggaa cagtttgcag ataaaaatct tagctatgat 2760 gaaagatcaa tcagcattat gaaggtggct caagcgaaac tgaaggaaat tggtccagat 2820 gacatgaata tggaagagta caagaagtgg catgaagatt atagtttgtt ccgaaaagtg 2880 tctgtgtatc tcctaacagg cctagaactc tatcaaaaag gaaagtacca agaggcactt 2940 tcctacctgg tatatgccta ccagagcaat gctgccctgc tgatgaaggg gccccgccgg 3000 ggggtcaaag aatccgtgat tgctttatac cgaagaaaat gccttctgga gctgaatgcc 3060 aaagcagctt ctctttttga aacaaatgat gatcactccg taactgaggg cattaatgtg 3120 atgaatgaac tgatcatccc ctgcattcac cttatcatta ataatgacat ttccaaggat 3180 gatctggatg ccattgaggt catgagaaac cattggtgct cttaccttgg gcaagatatt 3240 gcagaaaatc tgcagctgtg cctaggggag tttctaccca gacttctaga tccttctgca 3300 gaaatcatcg tcttgaaaga gcctccaact attcgaccca attctcccta tgacctatgt 3360 agccgatttg cagctgtcat ggagtcaatt cagggagttt caactgtgac agtgaaataa 3420 7 2082 DNA Homo sapiens CDS (115)...(1518) 7 cactagtaac gccgccatgt gctggaattc gcccttctcg ggaagcgcgc cattgtgttg 60 gtacccggga attcgcggcc gcgtcgacgc ccgccggggc tctccagctt cgcc atg 117 Met 1 ccg ccg tgg ggc gcc gcc ctc gcg ctc atc ttg gcc gtg ctc gcc ctt 165 Pro Pro Trp Gly Ala Ala Leu Ala Leu Ile Leu Ala Val Leu Ala Leu 5 10 15 ctc ggc ctg ctc ggc ccg cgg ctc cgg gga ccc tgg ggg cgc gcc gtc 213 Leu Gly Leu Leu Gly Pro Arg Leu Arg Gly Pro Trp Gly Arg Ala Val 20 25 30 gga gag agg acc ctg ccg ggg gcc caa gac cga gac gac ggg gag gag 261 Gly Glu Arg Thr Leu Pro Gly Ala Gln Asp Arg Asp Asp Gly Glu Glu 35 40 45 gcg gac ggc gga ggc ccg gcg gac cag ttc agc gac ggg cgc gag cca 309 Ala Asp Gly Gly Gly Pro Ala Asp Gln Phe Ser Asp Gly Arg Glu Pro 50 55 60 65 ctg ccg gga ggg tgc agc ctt gtt tgc aag ccg tcg gcc ctg gcc cag 357 Leu Pro Gly Gly Cys Ser Leu Val Cys Lys Pro Ser Ala Leu Ala Gln 70 75 80 tgc ctg ctg cgc gcc ctg cgg cgc tca gag gcg ctg gag gcc ggc ccg 405 Cys Leu Leu Arg Ala Leu Arg Arg Ser Glu Ala Leu Glu Ala Gly Pro 85 90 95 cgc tcc tgg ttc tcc ggg ccc cac ctg cag acc ctc tgc cac ttc gtc 453 Arg Ser Trp Phe Ser Gly Pro His Leu Gln Thr Leu Cys His Phe Val 100 105 110 ctg ccc gta gcg cct ggg cct gag ctg gcc cgg gag tac ctg cag ttg 501 Leu Pro Val Ala Pro Gly Pro Glu Leu Ala Arg Glu Tyr Leu Gln Leu 115 120 125 gcg gac gat ggg cta gtg gcc ctg gac tgg gtg gta gga cct tgt gtt 549 Ala Asp Asp Gly Leu Val Ala Leu Asp Trp Val Val Gly Pro Cys Val 130 135 140 145 cgg ggc cgc cgg atc acc agc gcc ggg ggc ctt cct gcg gtg ctt ctg 597 Arg Gly Arg Arg Ile Thr Ser Ala Gly Gly Leu Pro Ala Val Leu Leu 150 155 160 gtg atc ccc aat gcg tgg ggt cgc ctc acc cgc aac gtg ctc ggc ctt 645 Val Ile Pro Asn Ala Trp Gly Arg Leu Thr Arg Asn Val Leu Gly Leu 165 170 175 tgc ttg ctc gcc ctg gag cgc ggc tac tac ccg gtc atc ttc cat cgc 693 Cys Leu Leu Ala Leu Glu Arg Gly Tyr Tyr Pro Val Ile Phe His Arg 180 185 190 cgc ggc cac cac ggt tgc cca ctg gtc agc ccc cgg ctg cag cct ttc 741 Arg Gly His His Gly Cys Pro Leu Val Ser Pro Arg Leu Gln Pro Phe 195 200 205 ggg gac ccg tcc gac ctc aag gag gcg gtc aca tac atc cgc ttc cga 789 Gly Asp Pro Ser Asp Leu Lys Glu Ala Val Thr Tyr Ile Arg Phe Arg 210 215 220 225 cac ccg gcg gcg ccg ctg ttc gcg gtg agc gaa ggc tcg ggc tcg gcg 837 His Pro Ala Ala Pro Leu Phe Ala Val Ser Glu Gly Ser Gly Ser Ala 230 235 240 ctg ctc ctg tcc tac ctg ggc gag tgc ggc tcc tcc agc tac gtg aca 885 Leu Leu Leu Ser Tyr Leu Gly Glu Cys Gly Ser Ser Ser Tyr Val Thr 245 250 255 ggc gcc gcc tgc atc tcg ccc gtg ctg cgc tgc cga gag tgg ttc gag 933 Gly Ala Ala Cys Ile Ser Pro Val Leu Arg Cys Arg Glu Trp Phe Glu 260 265 270 gcc ggc ctg ccc tgg ccc tac gag cgg ggc ttt ctg ctc cac cag aag 981 Ala Gly Leu Pro Trp Pro Tyr Glu Arg Gly Phe Leu Leu His Gln Lys 275 280 285 atc gcc ctc agc agg tat gcc aca gcc ctg gag gac act gtg gac acc 1029 Ile Ala Leu Ser Arg Tyr Ala Thr Ala Leu Glu Asp Thr Val Asp Thr 290 295 300 305 agc aga ctg ttc agg agc cgt tcc ctt cga gag ttt gag gag gct ctc 1077 Ser Arg Leu Phe Arg Ser Arg Ser Leu Arg Glu Phe Glu Glu Ala Leu 310 315 320 ttc tgc cac acc aaa agc ttc ccc atc agc tgg gat gcc tac tgg gac 1125 Phe Cys His Thr Lys Ser Phe Pro Ile Ser Trp Asp Ala Tyr Trp Asp 325 330 335 cgc aac gac ccg ctc cgg gat gtc gat gag gca gcc gtg cct gtg ctg 1173 Arg Asn Asp Pro Leu Arg Asp Val Asp Glu Ala Ala Val Pro Val Leu 340 345 350 tgt atc tgc agt gct gac gac ccc gtg tgt gga ccc cca gac cac act 1221 Cys Ile Cys Ser Ala Asp Asp Pro Val Cys Gly Pro Pro Asp His Thr 355 360 365 ctg aca act gaa ctc ttc cac agc aac ccc tac ttc ttc ctc ctg ctc 1269 Leu Thr Thr Glu Leu Phe His Ser Asn Pro Tyr Phe Phe Leu Leu Leu 370 375 380 385 agt cgc cac gga ggc cac tgt ggc ttc ctg cgc cag gag ccc ttg cca 1317 Ser Arg His Gly Gly His Cys Gly Phe Leu Arg Gln Glu Pro Leu Pro 390 395 400 gcc tgg agc cat gag gtc atc ttg gag tcc ttc cgg gcc ttg act gag 1365 Ala Trp Ser His Glu Val Ile Leu Glu Ser Phe Arg Ala Leu Thr Glu 405 410 415 ttc ttc cga acg gag gag agg att aaa ggg ctg agc agg cac aga gct 1413 Phe Phe Arg Thr Glu Glu Arg Ile Lys Gly Leu Ser Arg His Arg Ala 420 425 430 tcc ttc ctt ggg ggc cgt cgt cgt ggg gga gcc ttg cag agg cgg gaa 1461 Ser Phe Leu Gly Gly Arg Arg Arg Gly Gly Ala Leu Gln Arg Arg Glu 435 440 445 gtc tct tcc tct tcc aac ctg gag gag atc ttt aac tgg aag cga tca 1509 Val Ser Ser Ser Ser Asn Leu Glu Glu Ile Phe Asn Trp Lys Arg Ser 450 455 460 465 tac aca agg tgagagacct ggcctgagaa cccccaagtc ctgcaaagaa 1558 Tyr Thr Arg aaacagagct gggcaagggg gagtcctgga aagatggggc ggactgaaca gagggagctc 1618 cagctctgtg ctcctcattc agtccctctc tcttaaattg gtgccttgaa agagaaggaa 1678 cgtcctgcga gcctgcactc acttcatcct cagcagaact cctgcctggc ctctgctcaa 1738 catatcccta ctcatccggt cagcagcggc gcgttccagt cactgtcacc tgtcactgac 1798 atcacaagcc aaaggatagc actttttcaa tccatggact caggagaaaa tgccctctta 1858 ctggcagtgg ctagagggat gagacgtttg tgtatgtcac tgggcagtga ccccgattct 1918 caagctggag ccatttgatg tcatgaggac aggatgtttg tgtctcggcc ccacttccct 1978 catttgctct gtggttgtgg cgccctgctt tgaccgaatg ctctggcaac tgcggcagca 2038 ggcttgtgtg tgtgagaagg gcggcagagg cagtggggct ggct 2082 8 468 PRT Homo sapiens 8 Met Pro Pro Trp Gly Ala Ala Leu Ala Leu Ile Leu Ala Val Leu Ala 1 5 10 15 Leu Leu Gly Leu Leu Gly Pro Arg Leu Arg Gly Pro Trp Gly Arg Ala 20 25 30 Val Gly Glu Arg Thr Leu Pro Gly Ala Gln Asp Arg Asp Asp Gly Glu 35 40 45 Glu Ala Asp Gly Gly Gly Pro Ala Asp Gln Phe Ser Asp Gly Arg Glu 50 55 60 Pro Leu Pro Gly Gly Cys Ser Leu Val Cys Lys Pro Ser Ala Leu Ala 65 70 75 80 Gln Cys Leu Leu Arg Ala Leu Arg Arg Ser Glu Ala Leu Glu Ala Gly 85 90 95 Pro Arg Ser Trp Phe Ser Gly Pro His Leu Gln Thr Leu Cys His Phe 100 105 110 Val Leu Pro Val Ala Pro Gly Pro Glu Leu Ala Arg Glu Tyr Leu Gln 115 120 125 Leu Ala Asp Asp Gly Leu Val Ala Leu Asp Trp Val Val Gly Pro Cys 130 135 140 Val Arg Gly Arg Arg Ile Thr Ser Ala Gly Gly Leu Pro Ala Val Leu 145 150 155 160 Leu Val Ile Pro Asn Ala Trp Gly Arg Leu Thr Arg Asn Val Leu Gly 165 170 175 Leu Cys Leu Leu Ala Leu Glu Arg Gly Tyr Tyr Pro Val Ile Phe His 180 185 190 Arg Arg Gly His His Gly Cys Pro Leu Val Ser Pro Arg Leu Gln Pro 195 200 205 Phe Gly Asp Pro Ser Asp Leu Lys Glu Ala Val Thr Tyr Ile Arg Phe 210 215 220 Arg His Pro Ala Ala Pro Leu Phe Ala Val Ser Glu Gly Ser Gly Ser 225 230 235 240 Ala Leu Leu Leu Ser Tyr Leu Gly Glu Cys Gly Ser Ser Ser Tyr Val 245 250 255 Thr Gly Ala Ala Cys Ile Ser Pro Val Leu Arg Cys Arg Glu Trp Phe 260 265 270 Glu Ala Gly Leu Pro Trp Pro Tyr Glu Arg Gly Phe Leu Leu His Gln 275 280 285 Lys Ile Ala Leu Ser Arg Tyr Ala Thr Ala Leu Glu Asp Thr Val Asp 290 295 300 Thr Ser Arg Leu Phe Arg Ser Arg Ser Leu Arg Glu Phe Glu Glu Ala 305 310 315 320 Leu Phe Cys His Thr Lys Ser Phe Pro Ile Ser Trp Asp Ala Tyr Trp 325 330 335 Asp Arg Asn Asp Pro Leu Arg Asp Val Asp Glu Ala Ala Val Pro Val 340 345 350 Leu Cys Ile Cys Ser Ala Asp Asp Pro Val Cys Gly Pro Pro Asp His 355 360 365 Thr Leu Thr Thr Glu Leu Phe His Ser Asn Pro Tyr Phe Phe Leu Leu 370 375 380 Leu Ser Arg His Gly Gly His Cys Gly Phe Leu Arg Gln Glu Pro Leu 385 390 395 400 Pro Ala Trp Ser His Glu Val Ile Leu Glu Ser Phe Arg Ala Leu Thr 405 410 415 Glu Phe Phe Arg Thr Glu Glu Arg Ile Lys Gly Leu Ser Arg His Arg 420 425 430 Ala Ser Phe Leu Gly Gly Arg Arg Arg Gly Gly Ala Leu Gln Arg Arg 435 440 445 Glu Val Ser Ser Ser Ser Asn Leu Glu Glu Ile Phe Asn Trp Lys Arg 450 455 460 Ser Tyr Thr Arg 465 9 1407 DNA Homo sapiens 9 atgccgccgt ggggcgccgc cctcgcgctc atcttggccg tgctcgccct tctcggcctg 60 ctcggcccgc ggctccgggg accctggggg cgcgccgtcg gagagaggac cctgccgggg 120 gcccaagacc gagacgacgg ggaggaggcg gacggcggag gcccggcgga ccagttcagc 180 gacgggcgcg agccactgcc gggagggtgc agccttgttt gcaagccgtc ggccctggcc 240 cagtgcctgc tgcgcgccct gcggcgctca gaggcgctgg aggccggccc gcgctcctgg 300 ttctccgggc cccacctgca gaccctctgc cacttcgtcc tgcccgtagc gcctgggcct 360 gagctggccc gggagtacct gcagttggcg gacgatgggc tagtggccct ggactgggtg 420 gtaggacctt gtgttcgggg ccgccggatc accagcgccg ggggccttcc tgcggtgctt 480 ctggtgatcc ccaatgcgtg gggtcgcctc acccgcaacg tgctcggcct ttgcttgctc 540 gccctggagc gcggctacta cccggtcatc ttccatcgcc gcggccacca cggttgccca 600 ctggtcagcc cccggctgca gcctttcggg gacccgtccg acctcaagga ggcggtcaca 660 tacatccgct tccgacaccc ggcggcgccg ctgttcgcgg tgagcgaagg ctcgggctcg 720 gcgctgctcc tgtcctacct gggcgagtgc ggctcctcca gctacgtgac aggcgccgcc 780 tgcatctcgc ccgtgctgcg ctgccgagag tggttcgagg ccggcctgcc ctggccctac 840 gagcggggct ttctgctcca ccagaagatc gccctcagca ggtatgccac agccctggag 900 gacactgtgg acaccagcag actgttcagg agccgttccc ttcgagagtt tgaggaggct 960 ctcttctgcc acaccaaaag cttccccatc agctgggatg cctactggga ccgcaacgac 1020 ccgctccggg atgtcgatga ggcagccgtg cctgtgctgt gtatctgcag tgctgacgac 1080 cccgtgtgtg gacccccaga ccacactctg acaactgaac tcttccacag caacccctac 1140 ttcttcctcc tgctcagtcg ccacggaggc cactgtggct tcctgcgcca ggagcccttg 1200 ccagcctgga gccatgaggt catcttggag tccttccggg ccttgactga gttcttccga 1260 acggaggaga ggattaaagg gctgagcagg cacagagctt ccttccttgg gggccgtcgt 1320 cgtgggggag ccttgcagag gcgggaagtc tcttcctctt ccaacctgga ggagatcttt 1380 aactggaagc gatcatacac aaggtga 1407 10 32 PRT Artificial Sequence Consensus sequence 10 Thr Gly Leu Ile Asn Leu Gly Asn Thr Cys Tyr Met Asn Ser Val Leu 1 5 10 15 Gln Cys Leu Phe Ser Ile Pro Pro Leu Arg Asp Tyr Leu Leu Asp Ile 20 25 30 11 69 PRT Artificial Sequence Consensus sequence 11 Gly Pro Gly Lys Tyr Glu Leu Tyr Ala Val Val Val His Ser Gly Ser 1 5 10 15 Ser Leu Ser Gly Gly His Tyr Thr Ala Tyr Val Lys Lys Glu Asn Trp 20 25 30 Tyr Lys Phe Asp Asp Asp Lys Val Ser Arg Val Thr Glu Glu Glu Val 35 40 45 Leu Lys Glu Ser Gly Gly Glu Ser Gly Asp Thr Ser Ser Ala Tyr Ile 50 55 60 Leu Phe Tyr Glu Arg 65 12 41 PRT Artificial Sequence Consensus sequence 12 Glu Asp Glu Glu Lys Ile Glu Gln Leu Val Glu Met Gly Phe Asp Arg 1 5 10 15 Glu Glu Val Val Lys Ala Leu Arg Ala Thr Asn Gly Asn Gly Val Glu 20 25 30 Arg Ala Ala Glu Trp Leu Leu Ser His 35 40 13 18 PRT Artificial Sequence Consensus sequence 13 Glu Ser Glu Glu Glu Asp Leu Gln Leu Ala Leu Ala Leu Ser Leu Glu 1 5 10 15 Glu Ala 14 232 PRT Artificial Sequence Consensus sequence 14 Phe Arg Val Ile Leu Leu Asp Leu Arg Gly Phe Gly Glu Ser Ser Pro 1 5 10 15 Ser Asp Leu Ala Glu Tyr Arg Phe Asp Asp Leu Ala Glu Asp Leu Glu 20 25 30 Ala Leu Leu Asp Ala Leu Gly Leu Glu Lys Pro Val Ile Leu Val Gly 35 40 45 His Ser Met Gly Gly Ala Ile Ala Leu Ala Tyr Ala Ala Lys Tyr Pro 50 55 60 Glu Leu Arg Val Lys Ala Leu Val Leu Val Ser Pro Pro Leu Pro Ala 65 70 75 80 Gly Leu Ser Ser Asp Leu Phe Pro Arg Gln Gly Asn Leu Glu Gly Leu 85 90 95 Leu Leu Ala Asn Phe Arg Asn Arg Leu Ser Arg Ser Val Glu Ala Leu 100 105 110 Leu Gly Arg Ala Leu Lys Gln Phe Phe Leu Leu Gly Arg Pro Leu Val 115 120 125 Ser Asp Phe Leu Lys Gln Ala Glu Asp Trp Leu Ser Ser Leu Ile Arg 130 135 140 Gln Gly Glu Asp Asp Gly Gly Asp Gly Leu Leu Gly Ala Ala Val Ala 145 150 155 160 Leu Gly Lys Leu Leu Gln Trp Asp Leu Ser Ala Leu Lys Asp Ile Lys 165 170 175 Val Pro Thr Leu Val Ile Trp Gly Thr Asp Asp Pro Leu Val Pro Leu 180 185 190 Asp Ala Ser Glu Lys Leu Ser Ala Leu Ile Pro Asn Ala Glu Val Val 195 200 205 Val Ile Asp Asp Ala Gly His Leu Ala Leu Leu Glu Lys Pro Glu Glu 210 215 220 Val Ala Glu Leu Ile Lys Phe Leu 225 230 15 341 PRT Artificial Sequence Consensus sequence 15 Pro Asn Phe Trp Leu Phe Asn Gly His Val Gln Thr Ile Trp Ala Ser 1 5 10 15 Phe Phe Arg Arg Lys Arg Cys Pro Thr Val Tyr Tyr Arg Arg Glu Ile 20 25 30 Leu Glu Leu Lys Asp Gly Gly Thr Val Thr Leu Asp Trp Met Glu Pro 35 40 45 Glu Gly Glu Asp Gln Asp Phe Asn Ser Asp Pro Asp Ser Pro Leu Val 50 55 60 Val Ile Leu His Gly Leu Thr Gly Gly Ser His Glu Pro Tyr Ile Arg 65 70 75 80 His Leu Val His Glu Leu Ala Arg Lys Arg Gly Trp Arg Cys Val Val 85 90 95 Leu Asn His Arg Gly Cys Gly Gly Ser Pro Ile Thr Thr Pro Arg Leu 100 105 110 Tyr Thr Ala Gly His Thr Glu Asp Ile Arg Glu Val Ile Glu His Leu 115 120 125 Lys Gln Arg Tyr Pro Glu Ala Pro Leu Tyr Ala Val Gly Phe Ser Leu 130 135 140 Gly Gly Asn Met Leu Thr Asn Tyr Leu Gly Glu Glu Gly Asp Asn Cys 145 150 155 160 Pro Leu Ser Ala Ala Val Thr Ile Cys Asn Pro Trp Asp Leu Glu Glu 165 170 175 Cys Ser Glu Ser Ile Glu Lys Gly Leu Met Ser Arg Arg Leu Tyr Asn 180 185 190 Arg Tyr Leu Thr Lys Asn Leu Lys Arg Met Val Gln Arg His Arg Asn 195 200 205 His Phe Glu Asp Ile Glu Lys Lys Ala Glu Tyr Asn Ala Glu Glu Ile 210 215 220 Asp Leu Glu Arg Leu Lys Lys Ala Arg Thr Ile Arg Glu Phe Asp Asp 225 230 235 240 Asn Ile Thr Ala Pro Met Tyr Gly Phe Lys Asp Ala Glu Asp Tyr Tyr 245 250 255 Arg Gln Ala Ser Ser Met Pro Tyr Leu Asp Asn Ile Arg Val Pro Leu 260 265 270 Leu Cys Ile Asn Ala Ala Asp Asp Pro Phe Met Pro Glu Glu Ala Ile 275 280 285 Pro Pro Asp Glu Ala Lys Gln Asn Pro Asn Val Val Leu Val Ile Thr 290 295 300 Ser His Gly Gly His Ile Gly Phe Ile Glu Gly Thr Trp Tyr Pro Ser 305 310 315 320 Gly Ser Gln Trp Leu Asp Gln Thr Ile Met Glu Tyr Leu Glu Ser Phe 325 330 335 Arg Thr Asn Arg Arg 340 16 19 PRT Artificial Sequence Signature pattern 16 Tyr Xaa Leu Xaa Xaa Xaa Xaa Xaa His Xaa Gly Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Gly His Tyr 17 16 PRT Homo sapiens 17 Gly Leu Thr Asn Leu Gly Ala Thr Cys Tyr Leu Ala Ser Thr Ile Gln 1 5 10 15 18 16 PRT Homo sapiens 18 Gly Leu Lys Asn Val Gly Asn Thr Cys Trp Phe Ser Ala Val Ile Gln 1 5 10 15 19 18 PRT Homo sapiens 19 Tyr Asp Leu Ile Gly Val Thr Val His Thr Gly Thr Ala Asp Gly Gly 1 5 10 15 His Tyr 20 18 PRT Homo sapiens 20 Tyr Arg Leu His Ala Val Leu Val His Glu Gly Gln Ala Asn Ala Gly 1 5 10 15 His Tyr 

What is claimed is:
 1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO:
 8. 2. The nucleic acid molecule of claim 1, further comprising vector nucleic acid sequences.
 3. The nucleic acid molecule of claim 1, further comprising nucleic acid sequences encoding a heterologous polypeptide.
 4. A host cell which contains the nucleic acid molecule of claim
 1. 5. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO:
 8. 6. The polypeptide of claim 5 further comprising heterologous amino acid sequences.
 7. An antibody or antigen-binding fragment thereof that selectively binds to a polypeptide of claim
 5. 8. A method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, the method comprising culturing the host cell of claim 4 under conditions in which the nucleic acid molecule is expressed.
 9. A method for detecting the presence of a polypeptide of claim 5 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 8; and b) determining whether the compound binds to the polypeptide in the sample.
 10. The method of claim 9, wherein the compound which binds to the polypeptide is an antibody.
 11. A kit comprising a compound which selectively binds to a polypeptide of claim 5 and instructions for use.
 12. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
 13. The method of claim 12, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
 14. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.
 15. A method for identifying a compound which binds to a polypeptide of claim 5 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 5 with a test compound; and b) determining whether the polypeptide binds to the test compound.
 16. A method for modulating the activity of a polypeptide of claim 5, comprising contacting a polypeptide or a cell expressing a polypeptide of claim 5 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
 17. A method of inhibiting aberrant activity of a 23479, 48120, or 46689-expressing cell, comprising contacting a 23479, 48120, or 46689-expressing cell with a compound that modulates the activity or expression of a polypeptide of claim 5, in an amount which is effective to reduce or inhibit the aberrant activity of the cell.
 18. The method of claim 17, wherein the compound is selected from the group consisting of a peptide, a phosphopeptide, a small organic molecule, and an antibody.
 19. The method of claim 17, wherein the cell is located in a cancerous or pre-cancerous tissue.
 20. A method of treating or preventing a disorder characterized by aberrant activity of a 23479, 48120, or 46689-expressing cell, in a subject, comprising: administering to the subject an effective amount of a compound that modulates the activity or expression of a nucleic acid molecule of claim 1, such that the aberrant activity of the 23479, 48120, or 46689-expressing cell is reduced or inhibited.
 21. A method of diagnosing a cellular proliferative or differentiative disorder in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to a nucleic acid molecule of claim 1; c) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample; and d) evaluating whether the amount of nucleic acid molecules in the sample as compared to a reference sample, wherein an increase in the amount of nucleic acid molecules in the sample compared to the reference sample is an indication that the subject has, or is at risk of having, a cellular proliferative or differentiative disorder.
 22. The method of claim 21, wherein the reference sample is a normal tissue of the same type as the sample tissue.
 23. The method of claim 22, wherein the tissue sample is obtained from the lung. 